Methods of treating glioblastoma

ABSTRACT

Disclosed herein are methods and compounds for treating glioblastoma with iniparib. Also disclosed herein is a method of selecting subjects having a glioblastoma for treatment based on a biomarker panel.

CROSS-REFERENCE

This application is a continuation of U.S. Application No. 17/152,506,filed on Jan. 19, 2021, which is a continuation of U.S. Application No.16/626,282, filed on Dec. 23, 2019, which is a U.S. National Phase ofInternational Application No. PCT/US2018/039103, filed on Jun. 22, 2018,which claims the benefit of U.S. Provisional Application No. 62/523,689,filed on Jun. 22, 2017, each of which is incorporated herein byreference in its entirety.

BACKGROUND OF THE DISCLOSURE

Cancer can develop in any tissue or organ at any age. The etiology ofcancer may not be clearly defined at times; however, mechanisms such asgenetic susceptibility, chromosome breakage disorders, viruses,environmental factors and immunologic disorders have all been linked toa malignant cell growth and transformation.

Worldwide, more than 10 million people are diagnosed with cancer everyyear and it is estimated that this number will grow to about 15 millionnew cases every year by 2020. Cancer causes six million deaths everyyear or about 12% of the deaths worldwide.

SUMMARY OF THE DISCLOSURE

Disclosed herein, in certain embodiments, are methods of treating asubject having glioblastoma. In some embodiments, also disclosed hereinare methods of selecting subjects having a glioblastoma for treatmentbased on a biomarker panel described herein. In additional embodiments,described herein are methods of monitoring the treatment progress basedon the expression level of biomarkers from the biomarker panel describedherein.

Disclosed herein, in certain embodiments, is a method of selecting asubject having glioblastoma or suspected of having glioblastoma fortreatment, the method comprising: (a) processing an extracted genomicDNA with a deaminating agent to generate a genomic DNA sample comprisingdeaminated nucleotides, wherein the extracted genomic DNA is obtainedfrom a biological sample from the subject having glioblastoma orsuspected of having glioblastoma; (b) generating a methylation profilecomprising the gene MGMT; (c) determining whether the subject has anMGMT promoter methylation; and (d) selecting subjects with an MGMTpromoter methylation for treatment with about 2 mg/kg to about 10 mg/kgof 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof;about 0 mg/m² to about 90 mg/m² of temozolomide; and radiation. In someembodiments, disclosed herein is a method of treating a subject havingglioblastoma characterized with an MGMT promoter methylation, comprisingadministering to the subject about 2 mg/kg to about 10 mg/kg of4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof; about0 mg/m² to about 90 mg/m² of temozolomide; and radiation, therebytreating the subject having glioblastoma characterized with an MGMTpromoter methylation, wherein the subject is determined to have the MGMTpromoter methylation by a) processing an extracted genomic DNA with adeaminating agent to generate a genomic DNA sample comprising deaminatednucleotides, wherein the extracted genomic DNA is obtained from abiological sample from the subject having glioblastoma or suspected ofhaving glioblastoma; b) generating a methylation profile comprising thegene MGMT; and determining whether the subject has an MGMT promotermethylation. In some embodiments, disclosed herein is a method oftreating a subject with a treatment regimen comprising4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof,wherein the subject is suffering from glioblastoma, the methodcomprising: determining whether the subject has an MGMT promotermethylation comprising: obtaining or having obtained a biological samplefrom the subject; and performing or having performed a methylation assayon the biological sample to determine if the subject has the MGMTpromoter methylation; and if the subject has an MGMT promotermethylation, then administering the treatment regimen comprising4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof to thesubject, and if the subject does not have an MGMT promoter methylation,then administering a first-line treatment for glioblastoma to thesubject, wherein a medium overall survival for the subject having anMGMT promoter methylation is extended following administration of thetreatment regimen comprising 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof than it would be if the first-linetreatment were administered; wherein the treatment regimen comprising4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereofcomprises: about 2 mg/kg to about 10 mg/kg of 4-iodo-3-nitrobenzamide ora salt, metabolite or prodrug thereof; about 0 mg/m² to about 90 mg/m²of temozolomide; and radiation. In some embodiments, the methylationprofile further comprises the methylation status of TP53, PTEN, or acombination thereof. In some embodiments, about 6 mg/kg to about 9 mg/kgof 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered to the subject. In some embodiments, about 7 mg/kg to about8.6 mg/kg of 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrugthereof is administered to the subject. In some embodiments, about 8mg/kg to about 8.6 mg/kg of 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof is administered to the subject. In someembodiments, about 8 mg/kg of 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof is administered to the subject. In someembodiments, the 4-iodo-3-nitrobenzamide or a salt, metabolite orprodrug thereof is administered to the subject once per day. In someembodiments, the 4-iodo-3-nitrobenzamide or a salt, metabolite orprodrug thereof is administered to the subject for about twice a week.In some embodiments, the 4-iodo-3-nitrobenzamide or a salt, metaboliteor prodrug thereof is administered to the subject for about four, fiveor six weeks. In some embodiments, the 4-iodo-3-nitrobenzamide or asalt, metabolite or prodrug thereof is administered to the subject forabout six weeks. In some embodiments, about 70 mg/m² to about 80 mg/m²of temozolomide is administered to the subject. In some embodiments,about 75 mg/m² of temozolomide is administered to the subject. In someembodiments, about 0 mg/m² to about 70 mg/m² of temozolomide isadministered to the subject. In some embodiments, about 5 mg/m² oftemozolomide is administered to the subject. In some embodiments, about10 mg/m² of temozolomide is administered to the subject. In someembodiments, about 15 mg/m² of temozolomide is administered to thesubject. In some embodiments, about 20 mg/m² of temozolomide isadministered to the subject. In some embodiments, temozolomide is notadministered to the subject. In some embodiments, temozolomide isadministered to the subject daily. In some embodiments, temozolomide isadministered to the subject for about four, five or six weeks. In someembodiments, temozolomide is administered to the subject for about sixweeks. In some embodiments, about 60 Gy of radiation is administered tothe subject over the course of about four, five, or six weeks. In someembodiments, about 60 Gy of radiation is administered to the subjectover the course of about six weeks. In some embodiments, upon completionof about six weeks of treatment with a combination of4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof,temozolomide and radiation, the subject receives a treatment break ofabout four weeks. In some embodiments, the method further comprises amaintenance regimen. In some embodiments, the maintenance regimencomprises about 8.6 mg/kg of 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof, and about 150 mg/m² to about 200 mg/m² oftemozolomide. In some embodiments, the 4-iodo-3-nitrobenzamide or asalt, metabolite or prodrug thereof is administered to the subject onceper day. In some embodiments, the 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof is administered to the subject for abouttwice a week. In some embodiments, the 4-iodo-3-nitrobenzamide or asalt, metabolite or prodrug thereof is administered to the subject forabout 1-6 cycles. In some embodiments, the temozolomide is administeredto the subject on Days 1-5 of each cycle. In some embodiments, thetemozolomide is administered to the subject for about 1-6 cycles. Insome embodiments, each cycle comprises about 28 days. In someembodiments, the 4-iodo-3-nitrobenzamide or a metabolite thereof isformulated for parenteral administration. In some embodiments, theparenteral administration comprises intravenous, intra-arterial,intracranial, intracerebral, intracerebroventricular, or intrathecaladministration. In some embodiments, the 4-iodo-3-nitrobenzamide or asalt, metabolite or prodrug thereof is formulated as an injection. Insome embodiments, the 4-iodo-3-nitrobenzamide or a salt, metabolite orprodrug thereof is formulated as an infusion. In some embodiments, theglioblastoma is a primary glioblastoma. In some embodiments, theglioblastoma is a secondary tumor. In some embodiments, the subject hasa grade III or grade IV glioblastoma. In some embodiments, thecombination of 4-iodo-3-nitrobenzamide or a metabolite thereof,radiation, and optionally temozolomide extends the median overallsurvival. In some embodiments, the median overall survival is extendedto about 16 to about 24 months. In some embodiments, the median overallsurvival is extended to about 21 months. In some embodiments, thecombination of 4-iodo-3-nitrobenzamide or a metabolite thereof,radiation, and optionally temozolomide reduces the hazard rate of death.In some embodiments, the hazard rate of death is reduced from about 0.6to about 0.42.

Disclosed herein, in certain embodiments, is a method of treatingglioblastoma in a subject in need thereof, comprising administering tothe subject: about 8 mg/kg of 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof; about 75 mg/m² of temozolomide; andradiation.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the disclosure are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present disclosure will be obtained by reference tothe following detailed description that sets forth illustrativeembodiments, in which the principles of the disclosure are utilized, andthe accompanying drawings of which:

FIG. 1 illustrates an exemplary Phase I treatment schema.

FIG. 2 illustrates an exemplary Phase II treatment schema.

FIG. 3 shows an overall survival analysis from the Phase II trialdescribed herein.

FIG. 4 illustrates an overall survival by MGMT status from the Phase IItrial described herein.

FIG. 5 illustrates an increase in the percentage of patients with 2 yearsurvival and 3 year survival.

DETAILED DESCRIPTION OF THE DISCLOSURE

Glioblastomas, or glioblastoma multiforme (GBM), are tumors that arisefrom astrocytes or the star-shaped cells that make up the “glue-like,”or supportive tissue of the brain. Glioblastoma is fast-growing, and insome cases, it is the most common primary tumor of the central nervoussystem in adults. In some cases, glioblastoma is further classified intoprimary glioblastoma (or de novo glioblastoma) or secondary tumor. Inadditional cases, glioblastoma is divided into grade I, grade II, gradeIII and grade IV glioblastoma.

Glioblastoma is considered one of the more difficult cancers to treat,due to the heterogeneity at the cellular, molecular, biological andgenetic levels. For example, glioblastomas are characterized by extentof infiltration into the brain parenchyma, marked angiogenesis,intrinsic resistance to apoptosis and genomic instability (Furnari, etal., “Malignant astrocytic glioma: genetics, biology, and paths totreatment.” Genes Dev (2007) 21: 2683-2710). Furthermore, due to thetumor make-up comprising a diverse array of different cell types, andthe presence of heterogeneity at both transcriptional and genomiclevels, resistance to treatment and recurrence are elevated as comparedto other cancers. Khosla, D. “Concurrent therapy to enhanceradiotherapeutic outcomes in glioblastoma,” Ann Transl Med (2016) 4(3):54.

In some instances, treatments for glioblastoma comprise surgery,chemotherpay, radiation, or a combination thereof. In some cases,targeted therapy such as PDZ1i (113B7) which inhibits MDA09/Synteninactivity, rindopepimut (Celldex), and DCVax® (Northwest Biotherapeutics)are also contemplated.

In some embodiments, described herein is a method for treatingglioblastoma with a nitrobenzamide compound. In some embodiments, thenitrobenzamide compound is encompassed by Formula (I):

wherein R₁, R₂, R₃, R₄, and R₅ are, independently selected from thegroup consisting of hydrogen, hydroxy, amino, nitro, iodo, (C₁-C₆)alkyl, (C₁-C₆) alkoxy, (C₃-C₇) cycloalkyl, and phenyl, wherein at leasttwo of the five R₁, R₂, R₃, R₄, and R₅ substituents are always hydrogen,at least one of the five substituents are always nitro, and at least onesubstituent positioned adjacent to a nitro is always iodo, andpharmaceutically acceptable salts, solvates, isomers, tautomers,metabolites, analogs, or prodrugs thereof. R₁, R₂, R₃, R₄, and R₅ canalso be a halide such as chloro, fluoro, or bromo.

In some embodiments, the nitrobenzamide compound is4-iodo-3-nitrobenzamide (also known as iniparib and BSI201). In someinstances, 4-iodo-3-nitrobenzamide has the structure

In some embodiments, the nitrobenzamide compound is a compound describedin U.S. Pat. No 5,464,871.

In some embodiments, disclosed herein is a method of treatingglioblastoma with a combination of a nitrobenzamide compound describedsupra, temozolomide, and radiation. In some instances, thenitrobenzamide compound is a compound encompassed by Formula (I). Insome instances, the nitrobenzamide compound is 4-iodo-3-nitrobenzamideor a salt, metabolite or prodrug thereof.

In some embodiments, also disclosed herein is a method of treatingglioblastoma in a subject in need thereof, comprising administering tothe subject: about 8 mg/kg of 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof; about 75 mg/m² of temozolomide; andradiation.

In some embodiments, additionally disclosed herein is a method ofselecting a subject having glioblastoma or suspected of havingglioblastoma for treatment, the method comprising: (a) processing anextracted genomic DNA with a deaminating agent to generate a genomic DNAsample comprising deaminated nucleotides, wherein the extracted genomicDNA is obtained from a biological sample from the subject havingglioblastoma or suspected of having glioblastoma; (b) generating amethylation profile comprising the gene MGMT; (c) determining whetherthe subject has an MGMT promoter methylation; and (d) selecting subjectswith an MGMT promoter methylation for treatment with about 2 mg/kg toabout 10 mg/kg of 4-iodo-3-nitrobenzamide or a salt, metabolite orprodrug thereof; about 0 mg/m² to about 90 mg/m² of temozolomide; andradiation.

In some embodiments, disclosed herein is a method of treating a subjecthaving glioblastoma characterized with an MGMT promoter methylation,comprising administering to the subject about 2 mg/kg to about 10 mg/kgof 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof;about 0 mg/m² to about 90 mg/m² of temozolomide; and radiation, therebytreating the subject having glioblastoma characterized with an MGMTpromoter methylation, wherein the subject is determined to have the MGMTpromoter methylation by a) processing an extracted genomic DNA with adeaminating agent to generate a genomic DNA sample comprising deaminatednucleotides, wherein the extracted genomic DNA is obtained from abiological sample from the subject having glioblastoma or suspected ofhaving glioblastoma; b) generating a methylation profile comprising thegene MGMT, and determining whether the subject has an MGMT promotermethylation.

In some embodiments, disclosed herein is a method of treating a subjectwith a treatment regimen comprising 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof, wherein the subject is suffering fromglioblastoma, the method comprising: determining whether the subject hasan MGMT promoter methylation comprising: obtaining or having obtained abiological sample from the subject; and performing or having performed amethylation assay on the biological sample to determine if the subjecthas the MGMT promoter methylation; and if the subject has an MGMTpromoter methylation, then administering the treatment regimencomprising 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrugthereof to the subject, and if the subject does not have an MGMTpromoter methylation, then administering a first-line treatment forglioblastoma to the subject, wherein a medium overall survival for thesubject having an MGMT promoter methylation is extended followingadministration of the treatment regimen comprising4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof than itwould be if the first-line treatment were administered; wherein thetreatment regimen comprising 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof comprises: about 2 mg/kg to about 10 mg/kgof 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof;about 0 mg/m² to about 90 mg/m² of temozolomide; and radiation.

0⁶-alkylguanine-DNA alkyltransferase (also known as AGT, AGAT or MGMT)is a protein involved in repairing O⁶-methylguanine to guanine andprevents mismatch and additional errors during DNA replication andtranscription. The MGMT protein is encoded by theO⁶-methylguanin-DNA-methyltransferase (MGMT) gene. In some instances,promoter methylation silences the MGMT gene. In such cases, the presenceof methylation at the promoter region of the MGMT gene in a subjecthaving glioblastoma is correlated with improved response outcome andlonger overall survival relative to the response outcome of a subjecthaving glioblastoma without the presence of methylation at the promoterregion of the MGMT gene.

In some instances, the methylation profile further comprises themethylation status of TP53, PTEN, or a combination thereof. In somecases, the promoter region of TP53 and PTEN genes are methylated. Insome cases, the methylation profile comprises the promoter methylationstatus of TP53, PTEN, or a combination thereof.

In some embodiments, the method further comprises determining thepresence of a mutation in IDH1, IDH2, TP53, PTEN, or a combinationthereof. In some instances, a mutation in the IDH1 gene correlates to amutation at residue Arg-132 of the IDH1 protein (GenBank: CAG38738.1).In some cases, the mutation at position 132 is from Arg-132 to His, Ser,Cys, Gly, Val or Leu. In some instances, a mutation in the IDH2 genecorrelates to a mutation at residue Arg-172 of the IDH2 protein(UniProtKB/Swiss-Prot: P48735.2). In some cases, the mutation atposition 172 is from Arg-172 to Lys, Met or Gly. In some instances, oneor more mutations are present in the TP53 gene. In some instances, oneor more mutations are present in the PTEN gene. In some cases, the oneor more mutations in the PTEN gene is located in exon 2, 3, 4, 5, 6, 7,or a combination thereof.

In some instances, the method comprises determining the presence of thepromoter methylation of MGMT and the presence of a mutation in IDH1,IDH2, TP 5 3, PTEN, or a combination thereof. In some cases, the methodcomprises determining the presence of the promoter methylation of MGMTand the presence of a mutation in TP53, PTEN, or a combination thereof.In other cases, the method comprises determining the presence of thepromoter methylation of MGMT and the presence of a mutation in IDH1,IDH2, or a combination thereof.

In some embodiments, 4-iodo-3-nitrobenzamide or a salt, metabolite orprodrug thereof is administered from about 2 mg/kg to about 200 mg/kg.In some instances, 4-iodo-3-nitrobenzamide or a salt, metabolite orprodrug thereof is administered from about 2 mg/kg to about 150 mg/kg,from about 2 mg/kg to about 100 mg/kg, or from about 2 mg/kg to about 60mg/kg. In some instances, 4-iodo-3-nitrobenzamide or a salt, metaboliteor prodrug thereof is administered from about 5 mg/kg to about 150mg/kg, from about 5 mg/kg to about 100 mg/kg, or from about 5 mg/kg toabout 60 mg/kg. In some instances, 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof is administered at a range of about 5mg/kg to about 50 mg/kg, about 5 mg/kg to about 40 mg/kg, about 5 mg/kgto about 30 mg/kg, about 5 mg/kg to about 20 mg/kg, about 5 mg/kg toabout 10 mg/kg, about 6 mg/kg to about 60 mg/kg, about 6 mg/kg to about50 mg/kg, about 6 mg/kg to about 40 mg/kg, about 6 mg/kg to about 30mg/kg, about 6 mg/kg to about 20 mg/kg, about 6 mg/kg to about 10 mg/kg,about 6 mg/kg to about 9 mg/kg, about 7 mg/kg to about 60 mg/kg, about 7mg/kg to about 50 mg/kg, about 7 mg/kg to about 40 mg/kg, about 7 mg/kgto about 30 mg/kg, about 7 mg/kg to about 20 mg/kg, about 7 mg/kg toabout 10 mg/kg, about 7 mg/kg to about 9 mg/kg, about 7 mg/kg to about 8mg/kg, about 8 mg/kg to about 60 mg/kg, about 8 mg/kg to about 40 mg/kg,about 8 mg/kg to about 30 mg/kg, about 8 mg/kg to about 20 mg/kg, about8 mg/kg to about 10 mg/kg, about 8 mg/kg to about 9 mg/kg, or about 8mg/kg to about 8.6 mg/kg. In some embodiments, 4-iodo-3-nitrobenzamideor a salt, metabolite or prodrug thereof is administered at a range ofabout 6 mg/kg to about 40 mg/kg, about 6 mg/kg to about 30 mg/kg, about6 mg/kg to about 20 mg/kg, about 6 mg/kg to about 10 mg/kg, about 6mg/kg to about 9 mg/kg, about 7 mg/kg to about 30 mg/kg, about 7 mg/kgto about 20 mg/kg, about 7 mg/kg to about 9 mg/kg, about 7 mg/kg toabout 8 mg/kg, about 8 mg/kg to about 20 mg/kg, about 8 mg/kg to about 9mg/kg, or about 8 mg/kg to about 8.6 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at a range of about 5 mg/kg to about 40 mg/kg. In someembodiments, 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrugthereof is administered at a range of about 6 mg/kg to about 9 mg/kg. Insome embodiments, 4-iodo-3-nitrobenzamide or a salt, metabolite orprodrug thereof is administered at a range of about 6 mg/kg to about 8.6mg/kg. In some embodiments, 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof is administered at a range of about 6mg/kg to about 8 mg/kg. In some embodiments, 4-iodo-3-nitrobenzamide ora salt, metabolite or prodrug thereof is administered at a range ofabout 7 mg/kg to about 9 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at a range of about 7 mg/kg to about 8.6 mg/kg. In someembodiments, 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrugthereof is administered at a range of about 7 mg/kg to about 8 mg/kg. Insome embodiments, 4-iodo-3-nitrobenzamide or a salt, metabolite orprodrug thereof is administered at a range of about 8 mg/kg to about 9mg/kg. In some embodiments, 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof is administered at a range of about 8mg/kg to about 8.6 mg/kg.

In some embodiments, 4-iodo-3-nitrobenzamide or a salt, metabolite orprodrug thereof is administered at about 2 mg/kg, about 3 mg/kg, about 4mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about8.5 mg/kg, about 8.6 mg/kg, about 9 mg/kg, about 10 mg/kg, about 15mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 40 mg/kg,about 50 mg/kg, about 60 mg/kg, about 100 mg/kg, about 150 mg/kg, orabout 200 mg/kg. In some embodiments, 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof is administered at about 5 mg/kg, about 6mg/kg, about 7 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 8.6 mg/kg,about 9 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25mg/kg, about 30 mg/kg, or about 40 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 2 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 3 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 4 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 5 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 6 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 7 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 8 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 8.5 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 8.6 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 9 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 10 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 15 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 20 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 30 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 40 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 50 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 60 mg/kg.

In some embodiments, 4-iodo-3-nitrobenzamide or a salt, metabolite orprodrug thereof is administered to a subject at one or more dosingschedules. In some embodiments, the dosing schedule comprisesadministering to the subject 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof once per day, twice a week, three times aweek, four times a week, five times a week, daily, every other day, oncea month, twice a month, or every week. In some instances, the dosingschedule comprises administering to the subject 4-iodo-3-nitrobenzamideor a salt, metabolite or prodrug thereof once per day.

In some embodiments, the dosing schedule comprises administering to thesubject 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereofcontinuously for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or moreweeks. In some instances, the dosing schedule comprises administering tothe subject 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrugthereof continuously for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreweeks. In some instances, the dosing schedule comprises administering tothe subject 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrugthereof continuously for about 1, 2, 3, 4, 5, 6 or more weeks. In someinstances, the dosing schedule comprises administering to the subject4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereofcontinuously for about 5 weeks. In some instances, the dosing schedulecomprises administering to the subject 4-iodo-3-nitrobenzamide or asalt, metabolite or prodrug thereof continuously for about 6 weeks. Insome instances, the dosing schedule comprises administering to thesubject 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereofcontinuously for about 7 weeks. In some instances, the dosing schedulecomprises administering to the subject 4-iodo-3-nitrobenzamide or asalt, metabolite or prodrug thereof continuously for about 8 weeks. Insome instances, the dosing schedule comprises administering to thesubject 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereofcontinuously for about 9 weeks. In some instances, the dosing schedulecomprises administering to the subject 4-iodo-3-nitrobenzamide or asalt, metabolite or prodrug thereof continuously for about 10 weeks. Insome instances, a 5-week dosing schedule is considered as one cycle. Insome instances, a 6-week dosing schedule is considered as one cycle. Insome instances, a 7-week dosing schedule is considered as one cycle. Insome instances, a 8-week dosing schedule is considered as one cycle. Insome instances, a 9-week dosing schedule is considered as one cycle. Insome instances, a 10-week dosing schedule is considered as one cycle. Insome instances, a 11-week dosing schedule is considered as one cycle. Insome instances, a 12-week dosing schedule is considered as one cycle.

In some embodiments, the dosing schedule comprises administering to thesubject 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereofcontinuously for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24,36, 48, or more months. In some instances, the dosing schedule comprisesadministering to the subject 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof continuously for about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, or more months. In some instances, the dosingschedule comprises administering to the subject 4-iodo-3-nitrobenzamideor a salt, metabolite or prodrug thereof continuously for about 1, 2, 3,4, 5, 6 or more months.

In some embodiments, the dosing schedule comprises administering to thesubject 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereofcontinuously for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more treatmentcycles. In some instances, the dosing schedule comprises administeringto the subject 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrugthereof continuously for about 1, 2, 3, 4, 5, 6 or more treatmentcycles. In some instances, the dosing schedule comprises administeringto the subject 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrugthereof continuously for about 1, 2, 3, 4 or more treatment cycles. Insome instances, the dosing schedule comprises administering to thesubject 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereofcontinuously for about 1 or more cycles. In some instances, the dosingschedule comprises administering to the subject 4-iodo-3-nitrobenzamideor a salt, metabolite or prodrug thereof continuously for about 2 ormore cycles. In some instances, the dosing schedule comprisesadministering to the subject 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof continuously for about 3 or more cycles.In some instances, the dosing schedule comprises administering to thesubject 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereofcontinuously for about 4 or more cycles. In some instances, eachtreatment cycle is up to 28 days. In some cases, each treatment cycle isabout 28 days. In other instances, each treatment cycle is up to 5weeks. In other instances, each treatment cycle is about 5 weeks. Inother instances, each treatment cycle is up to 6 weeks. In otherinstances, each treatment cycle is about 6 weeks. In other instances,each treatment cycle is up to 7 weeks. In other instances, eachtreatment cycle is about 7 weeks. In other instances, each treatmentcycle is up to 8 weeks. In other instances, each treatment cycle isabout 8 weeks. In other instances, each treatment cycle is up to 9weeks. In other instances, each treatment cycle is about 9 weeks. Inother instances, each treatment cycle is up to 10 weeks. In otherinstances, each treatment cycle is about 10 weeks. In some instances, a11-week dosing schedule is considered as one cycle. In some instances, a12-week dosing schedule is considered as one cycle.

In some embodiments, the dosing schedule comprises administering to thesubject 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereofintermittently for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or moreweeks. In some instances, the dosing schedule comprises administering tothe subject 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrugthereof intermittently for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreweeks. In some instances, the dosing schedule comprises administering tothe subject 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrugthereof intermittently for about 1, 2, 3, 4, 5, 6 or more weeks. In someinstances, the dosing schedule comprises administering to the subject4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereofintermittently for about 5 weeks. In some instances, the dosing schedulecomprises administering to the subject 4-iodo-3-nitrobenzamide or asalt, metabolite or prodrug thereof intermittently for about 6 weeks. Insome instances, the dosing schedule comprises administering to thesubject 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereofintermittently for about 7 weeks. In some instances, the dosing schedulecomprises administering to the subject 4-iodo-3-nitrobenzamide or asalt, metabolite or prodrug thereof intermittently for about 8 weeks. Insome instances, the dosing schedule comprises administering to thesubject 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereofintermittently for about 9 weeks. In some instances, the dosing schedulecomprises administering to the subject 4-iodo-3-nitrobenzamide or asalt, metabolite or prodrug thereof intermittently for about 10 weeks.In some instances, a 5-week dosing schedule is considered as one cycle.In some instances, a 6-week dosing schedule is considered as one cycle.In some instances, a 7-week dosing schedule is considered as one cycle.In some instances, a 8-week dosing schedule is considered as one cycle.In some instances, a 9-week dosing schedule is considered as one cycle.In some instances, a 10-week dosing schedule is considered as one cycle.

In some embodiments, the dosing schedule comprises administering to thesubject 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereofintermittently for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24,36, 48, or more months. In some instances, the dosing schedule comprisesadministering to the subject 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof intermittently for about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, or more months. In some instances, the dosingschedule comprises administering to the subject 4-iodo-3-nitrobenzamideor a salt, metabolite or prodrug thereof intermittently for about 1, 2,3, 4, 5, 6 or more months.

In some embodiments, the dosing schedule comprises administering to thesubject 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereofintermittently for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more treatmentcycles. In some instances, the dosing schedule comprises administeringto the subject 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrugthereof intermittently for about 1, 2, 3, 4, 5, 6 or more treatmentcycles. In some instances, the dosing schedule comprises administeringto the subject 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrugthereof intermittently for about 1, 2, 3, 4 or more treatment cycles. Insome instances, the dosing schedule comprises administering to thesubject 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereofintermittently for about 1 or more cycles. In some instances, the dosingschedule comprises administering to the subject 4-iodo-3-nitrobenzamideor a salt, metabolite or prodrug thereof intermittently for about 2 ormore cycles. In some instances, the dosing schedule comprisesadministering to the subject 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof intermittently for about 3 or more cycles.In some instances, the dosing schedule comprises administering to thesubject 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereofintermittently for about 4 or more cycles. In some instances, the dosingschedule comprises administering to the subject 4-iodo-3-nitrobenzamideor a salt, metabolite or prodrug thereof intermittently for about 5 ormore cycles. In some instances, the dosing schedule comprisesadministering to the subject 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof intermittently for about 6 or more cycles.In some instances, each treatment cycle is up to 28 days. In some cases,each treatment cycle is about 28 days. In other instances, eachtreatment cycle is up to 5 weeks. In other instances, each treatmentcycle is about 5 weeks. In other instances, each treatment cycle is upto 6 weeks. In other instances, each treatment cycle is about 6 weeks.In other instances, each treatment cycle is up to 7 weeks. In otherinstances, each treatment cycle is about 7 weeks. In other instances,each treatment cycle is up to 8 weeks. In other instances, eachtreatment cycle is about 8 weeks. In other instances, each treatmentcycle is up to 9 weeks. In other instances, each treatment cycle isabout 9 weeks. In other instances, each treatment cycle is up to 10weeks. In other instances, each treatment cycle is about 10 weeks.

In some cases, temozolomide is administered to a subject at a dosingrange of 70 mg/m² to about 200 mg/m², about 70 mg/m² to about 80 mg/m²,or about 150 mg/m² to about 200 mg/m². In some cases, temozolomide isadministered to a subject at a dosing range of about 70 mg/m² to about80 mg/m². In some cases, temozolomide is administered to a subject at adosing range of about 150 mg/m² to about 200 mg/m². In some cases, thedosing range of about 150 mg/m² to about 200 mg/m² is administered tothe subject as a maintenance regimen.

In some cases, temozolomide is administered to a subject at a dosingrange of about 60 mg/m², about 65 mg/m², about 70 mg/m², about 75 mg/m²,about 80 mg/m², about 85 mg/m², about 90 mg/m², about 95 mg/m², or about100 mg/m². In some cases, temozolomide is administered to a subject at adose of about 60 mg/m². In some cases, temozolomide is administered to asubject at a dose of about 65 mg/m². In some cases, temozolomide isadministered to a subject at a dose of about 70 mg/m². In some cases,temozolomide is administered to a subject at a dose of about 75 mg/m².In some cases, temozolomide is administered to a subject at a dose ofabout 80 mg/m². In some cases, temozolomide is administered to a subjectat a dose of about 85 mg/m². In some cases, temozolomide is administeredto a subject at a dose of about 90 mg/m². In some cases, temozolomide isadministered to a subject at a dose of about 95 mg/m². In some cases,temozolomide is administered to a subject at a dose of about 100 mg/m².

In some cases, temozolomide is administered to a subject at a dosingrange of 0 mg/m² to about 90 mg/m², about 0 mg/m² to about 80 mg/m²,about 0 mg/m² to about 70 mg/m², about 10 mg/m² to about 80 mg/m², about10 mg/m² to about 70 mg/m², about 10 mg/m² to about 60 mg/m², about 20mg/m² to about 80 mg/m², about 20 mg/m² to about 70 mg/m², about 20mg/m² to about 60 mg/m², about 30 mg/m² to about 80 mg/m², about 30mg/m² to about 70 mg/m², or about 30 mg/m² to about 60 mg/m². In somecases, temozolomide is administered to a subject at a dosing range of 0mg/m² to about 70 mg/m². In some cases, temozolomide is administered toa subject at a dosing range of 10 mg/m² to about 70 mg/m². In somecases, temozolomide is administered to a subject at a dosing range of 20mg/m² to about 70 mg/m². In some cases, temozolomide is administered toa subject at a dosing range of 30 mg/m² to about 70 mg/m².

In some cases, temozolomide is administered to a subject at a dose ofabout 0 mg/m², 5 mg/m², 10 mg/m², 15 mg/m², 20 mg/m², 25 mg/m², 30mg/m², 35 mg/m², 40 mg/m², 50 mg/m², 60 mg/m², 70 mg/m², 80 mg/m², or 90mg/m². In some cases, temozolomide is administered to a subject at adose of about 0 mg/m². In some cases, temozolomide is administered to asubject at a dose of about 5 mg/m². In some cases, temozolomide isadministered to a subject at a dose of about 10 mg/m². In some cases,temozolomide is administered to a subject at a dose of about 15 mg/m².In some cases, temozolomide is administered to a subject at a dose ofabout 20 mg/m². In some cases, temozolomide is administered to a subjectat a dose of about 25 mg/m². In some cases, temozolomide is administeredto a subject at a dose of about 30 mg/m². In some cases, temozolomide isadministered to a subject at a dose of about 35 mg/m². In some cases,temozolomide is administered to a subject at a dose of about 40 mg/m².In some cases, temozolomide is administered to a subject at a dose ofabout 50 mg/m². In some cases, temozolomide is administered to a subjectat a dose of about 60 mg/m². In some cases, temozolomide is administeredto a subject at a dose of about 70 mg/m². In some cases, temozolomide isadministered to a subject at a dose of about 80 mg/m². In some cases,temozolomide is administered to a subject at a dose of about 90 mg/m².

In some instances, temozolomide is administered to the subject daily. Insome cases, temozolomide is administered to the subject for about four,five, or about six weeks.

In some embodiments, temozolomide is not administered to the subject. Insuch cases, the method comprises, for example, selecting a subjecthaving glioblastoma or suspected of having glioblastoma for treatment,which comprises processing an extracted genomic DNA with a deaminatingagent to generate a genomic DNA sample comprising deaminatednucleotides, wherein the extracted genomic DNA is obtained from abiological sample from the subject having glioblastoma or suspected ofhaving glioblastoma, generating a methylation profile comprising thegene MGMT, determining whether the subj ect has a MGMT promotermethylation, and administering to the subject about 2 mg/kg to about 10mg/kg of 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrugthereof and radiation.

In some cases, the total dose of radiation administered to a subject isup to 60 gray (Gy). In some cases, the total dose of radiationadministered to a subject is up to 20 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50Gy, 55 Gy, or 60 Gy. In some cases, the total dose of radiationadministered to a subject is up to 20 Gy. In some cases, the total doseof radiation administered to a subject is up to 30 Gy. In some cases,the total dose of radiation administered to a subject is up to 35 Gy. Insome cases, the total dose of radiation administered to a subject is upto 40 Gy. In some cases, the total dose of radiation administered to asubject is up to 45 Gy. In some cases, the total dose of radiationadministered to a subject is up to 50 Gy. In some cases, the total doseof radiation administered to a subject is up to 55 Gy. In some cases,the total dose of radiation administered to a subject is up to 60 Gy. Insome instances, the total radiation dose is the dose a subject receivesover the course of a treatment cycle. In some instances, the treatmentcycle is from 3 to 10 weeks. In some instances, the treatment cycle isfrom 4 to 10 weeks, from 5 to 10 weeks, from 6 to 10 weeks, from 7 to 10weeks, from 4 to 9 weeks, from 5 to 9 weeks, from 6 to 9 weeks, from 5to 8 weeks, or from 6 to 8 weeks. In some instances, the treatment cycleis about 3 weeks. In some instances, the treatment cycle is about 4weeks. In some instances, the treatment cycle is about 5 weeks. In someinstances, the treatment cycle is about 6 weeks. In some instances, thetreatment cycle is about 7 weeks. In some instances, the treatment cycleis about 8 weeks. In some instances, the treatment cycle is about 9weeks. In some instances, the treatment cycle is about 10 weeks.

In some embodiments, a combination of 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof; temozolomide; and radiation isadministered to a subject. In some embodiments, the combination isadministered during a treatment phase (also referred to as initiationphase or initiation cycle). In some instances, the treatment phasecomprises an administration phase and a rest phase. In some cases, theadministration phase comprises about two, three, four, five, six, seven,eight, nine, ten, or more weeks in which the combination of4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof;temozolomide; and radiation is administered to the subject. In someinstances, the rest phase comprises about three, four, five, six or moreweeks in which no treatment is administered to the subject. In someembodiments, 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrugthereof is administered at about 5 mg/kg, about 6 mg/kg, about 7 mg/kg,about 8 mg/kg, about 8.5 mg/kg, about 8.6 mg/kg, about 9 mg/kg, about 10mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg,about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 100 mg/kg, about150 mg/kg, or about 200 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8mg/kg, about 8.5 mg/kg, about 8.6 mg/kg, about 9 mg/kg, about 10 mg/kg,about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, or about40 mg/kg. In some embodiments, 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof is administered at about 5 mg/kg. In someembodiments, 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrugthereof is administered at about 6 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 7 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 8 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 8.5 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 8.6 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 9 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 10 mg/kg. In some instances,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered about once per day. In some cases, 4-iodo-3-nitrobenzamideor a salt, metabolite or prodrug thereof is administered about twice aweek. In additional cases, 4-iodo-3-nitrobenzamide or a salt, metaboliteor prodrug thereof is administered about once per day and twice a week.In some cases, temozolomide is administered to a subject at a dose ofabout 60 mg/m², about 65 mg/m², about 70 mg/m², about 75 mg/m², about 80mg/m², about 85 mg/m², about 90 mg/m², about 95 mg/m², or about 100mg/m². In some cases, temozolomide is administered to a subject at adose of about 60 mg/m². In some cases, temozolomide is administered to asubject at a dose of about 65 mg/m². In some cases, temozolomide isadministered to a subject at a dose of about 70 mg/m². In some cases,temozolomide is administered to a subject at a dose of about 75 mg/m².In some cases, temozolomide is administered to a subject at a dose ofabout 80 mg/m². In some cases, temozolomide is administered to a subjectat a dose of about 85 mg/m². In some cases, temozolomide is administeredto a subject at a dose of about 90 mg/m². In some cases, temozolomide isadministered to a subject at a dose of about 95 mg/m². In some cases,temozolomide is administered to a subject at a dose of about 100 mg/m².In some embodiments, temozolomide is administered daily. In some cases,the total dose of radiation administered to a subject is up to 60 gray(Gy). In some cases, the total dose of radiation administered to asubject is up to 20 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50 Gy, 55 Gy, or 60Gy. In some cases, the total dose of radiation administered to a subjectis up to 20 Gy. In some cases, the total dose of radiation administeredto a subject is up to 30 Gy. In some cases, the total dose of radiationadministered to a subject is up to 35 Gy. In some cases, the total doseof radiation administered to a subject is up to 40 Gy. In some cases,the total dose of radiation administered to a subject is up to 45 Gy. Insome cases, the total dose of radiation administered to a subject is upto 50 Gy. In some cases, the total dose of radiation administered to asubject is up to 55 Gy. In some cases, the total dose of radiationadministered to a subject is up to 60 Gy.

In some embodiments, a combination of 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof; temozolomide; and radiation isadministered to a subject during the treatment phase for about six weeksfollowed by about four weeks of rest phase (or no treatment). In someembodiments, 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrugthereof is administered at about 5 mg/kg, about 6 mg/kg, about 7 mg/kg,about 8 mg/kg, about 8.5 mg/kg, about 8.6 mg/kg, about 9 mg/kg, about 10mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg,about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 100 mg/kg, about150 mg/kg, or about 200 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8mg/kg, about 8.5 mg/kg, about 8.6 mg/kg, about 9 mg/kg, about 10 mg/kg,about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, or about40 mg/kg. In some embodiments, 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof is administered at about 5 mg/kg. In someembodiments, 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrugthereof is administered at about 6 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 7 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 8 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 8.5 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 8.6 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 9 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 10 mg/kg. In some instances,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered about once per day. In some cases, 4-iodo-3-nitrobenzamideor a salt, metabolite or prodrug thereof is administered about twice aweek. In additional cases, 4-iodo-3-nitrobenzamide or a salt, metaboliteor prodrug thereof is administered about once per day and twice a week.In some cases, temozolomide is administered to a subject at a dose ofabout 60 mg/m², about 65 mg/m², about 70 mg/m², about 75 mg/m², about 80mg/m², about 85 mg/m², about 90 mg/m², about 95 mg/m², or about 100mg/m². In some cases, temozolomide is administered to a subject at adose of about 60 mg/m². In some cases, temozolomide is administered to asubject at a dose of about 65 mg/m². In some cases, temozolomide isadministered to a subject at a dose of about 70 mg/m². In some cases,temozolomide is administered to a subject at a dose of about 75 mg/m².In some cases, temozolomide is administered to a subject at a dose ofabout 80 mg/m². In some cases, temozolomide is administered to a subjectat a dose of about 85 mg/m². In some cases, temozolomide is administeredto a subject at a dose of about 90 mg/m². In some cases, temozolomide isadministered to a subject at a dose of about 95 mg/m². In some cases,temozolomide is administered to a subject at a dose of about 100 mg/m².In some embodiments, temozolomide is administered daily. In some cases,the total dose of radiation administered to a subject is up to 60 gray(Gy). In some cases, the total dose of radiation administered to asubject is up to 20 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50 Gy, 55 Gy, or 60Gy. In some cases, the total dose of radiation administered to a subjectis up to 20 Gy. In some cases, the total dose of radiation administeredto a subject is up to 30 Gy. In some cases, the total dose of radiationadministered to a subject is up to 35 Gy. In some cases, the total doseof radiation administered to a subject is up to 40 Gy. In some cases,the total dose of radiation administered to a subject is up to 45 Gy. Insome cases, the total dose of radiation administered to a subject is upto 50 Gy. In some cases, the total dose of radiation administered to asubject is up to 55 Gy. In some cases, the total dose of radiationadministered to a subject is up to 60 Gy.

In some embodiments, a combination of 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof; temozolomide; and radiation isadministered to a subject during the treatment phase for about six weeksfollowed by about four weeks of rest phase (or no treatment). In someembodiments, 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrugthereof is administered at about 5 mg/kg, about 6 mg/kg, about 7 mg/kg,about 8 mg/kg, about 8.5 mg/kg, about 8.6 mg/kg, about 9 mg/kg, about 10mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg,about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 100 mg/kg, about150 mg/kg, or about 200 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8mg/kg, about 8.5 mg/kg, about 8.6 mg/kg, about 9 mg/kg, about 10 mg/kg,about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, or about40 mg/kg. In some embodiments, 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof is administered at about 5 mg/kg. In someembodiments, 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrugthereof is administered at about 6 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 7 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 8 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 8.5 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 8.6 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 9 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 10 mg/kg. In some cases, 4-iodo-3-nitrobenzamideor a salt, metabolite or prodrug thereof is administered about once perday and twice a week. In some cases, temozolomide is administered to asubject at a dose of about 60 mg/m², about 65 mg/m², about 70 mg/m²,about 75 mg/m², about 80 mg/m², about 85 mg/m², about 90 mg/m², about 95mg/m², or about 100 mg/m². In some cases, temozolomide is administeredto a subject at a dose of about 60 mg/m². In some cases, temozolomide isadministered to a subject at a dose of about 65 mg/m². In some cases,temozolomide is administered to a subject at a dose of about 70 mg/m².In some cases, temozolomide is administered to a subject at a dose ofabout 75 mg/m². In some cases, temozolomide is administered to a subjectat a dose of about 80 mg/m². In some cases, temozolomide is administeredto a subject at a dose of about 85 mg/m². In some cases, temozolomide isadministered to a subject at a dose of about 90 mg/m². In some cases,temozolomide is administered to a subject at a dose of about 95 mg/m².In some cases, temozolomide is administered to a subject at a dose ofabout 100 mg/m². In some embodiments, temozolomide is administereddaily. In some cases, the total dose of radiation administered to asubject is up to 60 gray (Gy). In some cases, the total dose ofradiation administered to a subject is up to 20 Gy, 30 Gy, 35 Gy, 40 Gy,45 Gy, 50 Gy, 55 Gy, or 60 Gy. In some cases, the total dose ofradiation administered to a subject is up to 20 Gy. In some cases, thetotal dose of radiation administered to a subject is up to 30 Gy. Insome cases, the total dose of radiation administered to a subject is upto 35 Gy. In some cases, the total dose of radiation administered to asubject is up to 40 Gy. In some cases, the total dose of radiationadministered to a subject is up to 45 Gy. In some cases, the total doseof radiation administered to a subject is up to 50 Gy. In some cases,the total dose of radiation administered to a subject is up to 55 Gy. Insome cases, the total dose of radiation administered to a subject is upto 60 Gy.

In some embodiments, a combination of 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof; and radiation is administered to asubject during the treatment phase for about six weeks followed by aboutfour weeks of rest phase (or no treatment). In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8mg/kg, about 8.5 mg/kg, about 8.6 mg/kg, about 9 mg/kg, about 10 mg/kg,about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 40mg/kg, about 50 mg/kg, about 60 mg/kg, about 100 mg/kg, about 150 mg/kg,or about 200 mg/kg. In some embodiments, 4-iodo-3-nitrobenzamide or asalt, metabolite or prodrug thereof is administered at about 5 mg/kg,about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 8.6mg/kg, about 9 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg,about 25 mg/kg, about 30 mg/kg, or about 40 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 5 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 6 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 7 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 8 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 8.5 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 8.6 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 9 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 10 mg/kg. In some instances,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered about once per day. In some cases, 4-iodo-3-nitrobenzamideor a salt, metabolite or prodrug thereof is administered about twice aweek. In additional cases, 4-iodo-3-nitrobenzamide or a salt, metaboliteor prodrug thereof is administered about once per day and twice a week.In some cases, the total dose of radiation administered to a subject isup to 60 gray (Gy). In some cases, the total dose of radiationadministered to a subject is up to 20 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50Gy, 55 Gy, or 60 Gy. In some cases, the total dose of radiationadministered to a subject is up to 20 Gy. In some cases, the total doseof radiation administered to a subject is up to 30 Gy. In some cases,the total dose of radiation administered to a subject is up to 35 Gy. Insome cases, the total dose of radiation administered to a subject is upto 40 Gy. In some cases, the total dose of radiation administered to asubject is up to 45 Gy. In some cases, the total dose of radiationadministered to a subject is up to 50 Gy. In some cases, the total doseof radiation administered to a subject is up to 55 Gy. In some cases,the total dose of radiation administered to a subject is up to 60 Gy.

In some embodiments, a combination of 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof; radiation, and an additionalchemotherapeutic agent is administered to a subject during the treatmentphase for about six weeks followed by about four weeks of rest phase (orno treatment). In some embodiments, 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof is administered at about 5 mg/kg, about 6mg/kg, about 7 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 8.6 mg/kg,about 9 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg,about 100 mg/kg, about 150 mg/kg, or about 200 mg/kg. In someembodiments, 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrugthereof is administered at about 5 mg/kg, about 6 mg/kg, about 7 mg/kg,about 8 mg/kg, about 8.5 mg/kg, about 8.6 mg/kg, about 9 mg/kg, about 10mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg,or about 40 mg/kg. In some embodiments, 4-iodo-3-nitrobenzamide or asalt, metabolite or prodrug thereof is administered at about 5 mg/kg. Insome embodiments, 4-iodo-3-nitrobenzamide or a salt, metabolite orprodrug thereof is administered at about 6 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 7 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 8 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 8.5 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 8.6 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 9 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 10 mg/kg. In some instances,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered about once per day. In some cases, 4-iodo-3-nitrobenzamideor a salt, metabolite or prodrug thereof is administered about twice aweek. In additional cases, 4-iodo-3-nitrobenzamide or a salt, metaboliteor prodrug thereof is administered about once per day and twice a week.In some cases, the total dose of radiation administered to a subject isup to 60 gray (Gy). In some cases, the total dose of radiationadministered to a subject is up to 20 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50Gy, 55 Gy, or 60 Gy. In some cases, the total dose of radiationadministered to a subject is up to 20 Gy. In some cases, the total doseof radiation administered to a subject is up to 30 Gy. In some cases,the total dose of radiation administered to a subject is up to 35 Gy. Insome cases, the total dose of radiation administered to a subject is upto 40 Gy. In some cases, the total dose of radiation administered to asubject is up to 45 Gy. In some cases, the total dose of radiationadministered to a subject is up to 50 Gy. In some cases, the total doseof radiation administered to a subject is up to 55 Gy. In some cases,the total dose of radiation administered to a subject is up to 60 Gy. Insome instances, the additional chemotherapeutic agent comprisesprocarbazine, carmustine (BCNU), iomustine (CCNU), or vincristine. Insome instances, the additional chemotherapeutic agent does not includetemozolomide.

In some embodiments, upon completion of a treatment phase or initiationcycle, a maintenance cycle is then initiated. In some instances, themaintenance cycle comprises about 1-10, 1-8, or 1-6 cycles. In somecases, a cycle is about 28 days. In some cases, a cycle is about 4weeks. In some embodiments, 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof is administered at about 5 mg/kg, about 6mg/kg, about 7 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 8.6 mg/kg,about 9 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg,about 100 mg/kg, about 150 mg/kg, or about 200 mg/kg. In someembodiments, 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrugthereof is administered at about 5 mg/kg, about 6 mg/kg, about 7 mg/kg,about 8 mg/kg, about 8.5 mg/kg, about 8.6 mg/kg, about 9 mg/kg, about 10mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg,or about 40 mg/kg. In some embodiments, 4-iodo-3-nitrobenzamide or asalt, metabolite or prodrug thereof is administered at about 5 mg/kg. Insome embodiments, 4-iodo-3-nitrobenzamide or a salt, metabolite orprodrug thereof is administered at about 6 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 7 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 8 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 8.5 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 8.6 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 9 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 10 mg/kg. In some instances,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered about once per day. In some cases, 4-iodo-3-nitrobenzamideor a salt, metabolite or prodrug thereof is administered about twice aweek. In additional cases, 4-iodo-3-nitrobenzamide or a salt, metaboliteor prodrug thereof is administered about once per day and twice a week.In some cases, temozolomide is administered to a subject at a dosingrange of 70 mg/m² to about 200 mg/m², about 70 mg/m² to about 80 mg/m²,or about 150 mg/m² to about 200 mg/m². In some cases, temozolomide isadministered to a subject at a dosing range of about 150 mg/m² to about200 mg/m². In some cases, temozolomide is administered on Days 1-5 ofeach cycle. In some cases, the administration of temozolomide isrepeated every 28 days.

In some instances, the maintenance cycle comprises about 1-6 cycles. Insome cases, a cycle is about 28 days. In some cases, a cycle is about 4weeks. In some embodiments, 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof is administered at about 5 mg/kg, about 6mg/kg, about 7 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 8.6 mg/kg,about 9 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg,about 100 mg/kg, about 150 mg/kg, or about 200 mg/kg. In someembodiments, 4-iodo-3-nitrobenzamide or a salt, metabolite or prodrugthereof is administered at about 5 mg/kg, about 6 mg/kg, about 7 mg/kg,about 8 mg/kg, about 8.5 mg/kg, about 8.6 mg/kg, about 9 mg/kg, about 10mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg,or about 40 mg/kg. In some embodiments, 4-iodo-3-nitrobenzamide or asalt, metabolite or prodrug thereof is administered at about 5 mg/kg. Insome embodiments, 4-iodo-3-nitrobenzamide or a salt, metabolite orprodrug thereof is administered at about 6 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 7 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 8 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 8.5 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 8.6 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 9 mg/kg. In some embodiments,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered at about 10 mg/kg. In some instances,4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof isadministered about once per day. In some cases, 4-iodo-3-nitrobenzamideor a salt, metabolite or prodrug thereof is administered about twice aweek. In additional cases, 4-iodo-3-nitrobenzamide or a salt, metaboliteor prodrug thereof is administered about once per day and twice a week.In some cases, temozolomide is administered to a subject at a dosingrange of 70 mg/m² to about 200 mg/m², about 70 mg/m² to about 80 mg/m²,or about 150 mg/m² to about 200 mg/m². In some cases, temozolomide isadministered to a subject at a dosing range of about 150 mg/m² to about200 mg/m². In some cases, temozolomide is administered on Days 1-5 ofeach cycle. In some cases, the administration of temozolomide isrepeated every 28 days.

In some embodiments, the method further comprises determining theexpression level of at least one gene selected from thioredoxinreductase 2 (TXNRD2), thioredoxin 2 (TXN2), methionine sulfoxidereductase B3 (MSRB3), methionine sulfoxide reductase A (MSRA), andglutathione transferase zeta 1 (GSTZI) by i) contacting at least onegene selected from TXNRD2, TXN2, MSRB3, MSRA, and GSTZI with a set ofprimers to produce amplified nucleic acids, wherein the at least onegene is isolated from a tumor sample obtained from the subject; and ii)determining the level of the amplified nucleic acids in the tumor samplerelative to a control; wherein an elevated expression of the genesindicate that the subject is responsive to the treatment.

In some embodiments, the level of at least one gene selected fromTXNRD2, TXN2, MSRB3 and MSRA is determined. In some cases, the level oftwo or more genes selected from TXNRD2, TXN2, MSRB3 and MSRA aredetermined. In some cases, the level of TXNRD2 is determined. In somecases, the level of TXN2 is determined. In some cases, the level ofMSRB3 is determined. In some cases, the level of MSRA is determined. Insome cases, the level of TXNRD2, TXN2, MSRB3 and MSRA are determined.

In some embodiments, the level of the amplified nucleic acids from atleast one gene selected from TXNRD2, TXN2, MSRB3, MSRA and GSTZIcorrelates to a decreased risk of disease progression.

In some embodiments, the method further comprises determining the levelof amplified nucleic acids from at least one gene selected from NAD(P)Hdehydrogenase quinone 2 (NQ02), glutathione S-transferase theta 2(GSTT2), glutathione S-transferase M3 (GSTM3), glutaredoxin (GLRX),selenoprotein O (SELO), paraoxonase 1 (PONI), glutathione S-transferaseomega 1 (GSTO1), glutaredoxin 3 (GLRX3), selenoprotein X 1 (SEPX1), andthioredoxin reductase 1 (TXNRDI) and comparing the level with a control.

In some embodiments, the level of at least one gene selected from NQO2,GSTT2, GSTM3, GLRX, GSTO1, GLRX3 and TXNRD1 is determined. In someinstances, the level of at least one gene selected from NQO2, GSTT2,GSTM3, GLRX, GSTO1 and GLRX3 is determined. In some instances, the levelof at least one gene selected from GSTT2, GSTM3, GLRX, GSTO1 and GLRX3is determined. In some instances, the level of at least one geneselected from GSTT2, GSTM3, and GSTO1 is determined. In some instances,the level of at least one gene selected from NQO2, SELO, PON1, SEPXI andTXNRD1 is determined. In some instances, the level of at least one geneselected from SELO, PON1, SEPXI and TXNRD1 is determined. In someinstances, the level of at least one gene selected from SELO, PON1 andSEPX1 is determined. In some instances, the level of NQO2 is determined.In some instances, the level of GSTT2 is determined. In some instances,the level of GSTM3 is determined. In some instances, the level of GLRXis determined. In some instances, the level of GSTO1 is determined. Insome instances, the level of GLRX3 is determined. In some instances, thelevel of TXNRD1 is determined.

In some cases, the treatment with 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof is discontinued if the level of amplifiednucleic acids is greater than the level in the control.

In some cases, the level of amplified nucleic acids greater than thelevel in the control correlates to an increased risk of diseaseprogression.

In some embodiments, a subject is diagnosed with a primary glioblastoma.In other embodiments, a subject is diagnosed with a secondary tumor. Insome cases, a subject is diagnosed with a grade I or grade IIglioblastoma. In other cases, a subject is diagnosed with a grade III ora grade IV glioblastoma. In some cases, the glioblastoma is ametastasized glioblastoma.

In some embodiments, the combination of 4-iodo-3-nitrobenzamide or ametabolite thereof, temozolomide and radiation extends the medianoverall survival. In some cases, the median overall survival is extendedto about 16 to about 24 months. In some cases, the median overallsurvival is extended to about 21 months.

In some embodiments, the combination of 4-iodo-3-nitrobenzamide or ametabolite thereof, and radiation extends the median overall survival.

In some embodiments, the hazard rate of death is reduced from about 0.6to about 0.42, with a combination of 4-iodo-3-nitrobenzamide or ametabolite thereof, temozolomide and radiation.

In some embodiments, the hazard rate of death is reduced with acombination of 4-iodo-3-nitrobenzamide or a metabolite thereof, andradiation.

In some embodiments, the combination of 4-iodo-3-nitrobenzamide or ametabolite thereof, radiation, and optionally temozolomide increases thecomplete response or partial response of a subject administered with thecombination, relative to a second subject who is not administered withthe combination.

In some embodiments, the combination of 4-iodo-3-nitrobenzamide or ametabolite thereof, radiation, and optionally temozolomide extends theprogression-free survival (PFS) of a subject administered with thecombination, relative to a second subject who is not administered withthe combination.

Samples and Detection Methods Samples

In some embodiments, a sample described herein is obtained from amammalian source. In some instances, the mammalian source compriseshuman and non-human primates. In other cases, the mammalian sourcecomprises a rodent (e.g., mouse, rat), cat, rabbit, dog, and the like.

In some cases, a sample described herein is a tissue sample. In somecases, the sample is a biopsy sample. In some cases, the sample is atumor sample, e.g., a tumor sample obtained from brain cancer, bladdercancer, breast cancer, colorectal cancer, lung cancer, or prostatecancer.

In some cases, a sample described herein is a liquid sample. In somecases, the liquid sample comprises blood and other liquid samples ofbiological origin (including, but not limited to, peripheral blood,sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva,bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen,breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid,cowper’s fluid or pre-ejaculatory fluid, female ejaculate, sweat, tears,cyst fluid, pleural and peritoneal fluid, pericardial fluid, ascites,lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum,vomit, vaginal secretions/flushing, synovial fluid, mucosal secretion,stool water, pancreatic juice, lavage fluids from sinus cavities,bronchopulmonary aspirates, blastocyl cavity fluid, or umbilical cordblood). In some embodiments, the sample is blood, a blood derivative ora blood fraction, e.g., serum or plasma. In some embodiments, the liquidsample also encompasses a sample that has been manipulated in any wayafter their procurement, such as by centrifugation, filtration,precipitation, dialysis, chromatography, treatment with reagents,washed, or enriched for certain cell populations.

In some embodiments, a sample described herein is a cell sample, e.g.,obtained from a tumor or a cancer cell line. In some instances, the cellsample is obtained from cells of brain cancer, bladder cancer, breastcancer, colorectal cancer, lung cancer, prostate cancer, large granularlymphocytic leukemia, T-cell acute lymphoblastic leukemia (T-ALL),T-cell prolymphocytic leukemia (T-PLL) or a melanoma.

In some instances, a sample described herein is a cell-free sample.

In some embodiments, the samples are obtained from the individual by anysuitable means of obtaining the sample using well-known and routineclinical methods. Procedures for obtaining fluid samples from anindividual are well known. For example, procedures for drawing andprocessing whole blood and lymph are well-known and can be employed toobtain a sample for use in the methods provided. Typically, forcollection of a blood sample, an anticoagulation agent (e.g., EDTA, orcitrate and heparin or CPD (citrate, phosphate, dextrose) or comparablesubstances) is added to the sample to prevent coagulation of the blood.In some examples, the blood sample is collected in a collection tubethat contains an amount of EDTA to prevent coagulation of the bloodsample.

In some embodiments, the collection of a sample from the subject isperformed at regular intervals, such as, for example, one day, two days,three days, four days, five days, six days, one week, two weeks, weeks,four weeks, one month, two months, three months, four months, fivemonths, six months, one year, daily, weekly, bimonthly, quarterly,biyearly or yearly.

In some embodiments, the collection of a sample is performed at apredetermined time or at regular intervals relative to treatment with4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof. Insome cases, the collection of a sample is performed at a predeterminedtime or at regular intervals relative to treatment with4-iodo-3-nitrobenzamide or a salt, metabolite or prodrug thereof and anadditional therapeutic agent described herein.

Detection Methods

In some embodiments, methods of detecting the expression level of one ormore biomarkers described herein include, but are not limited to,Western blots, Northern blots, Southern blots, enzyme-linkedimmunosorbent assay (ELISA), immunoprecipitation, immunofluorescence,radioimmunoassay, immunocytochemistry, nucleic acid hybridizationtechniques, nucleic acid reverse transcription methods, nucleic acidamplification methods, or a combination thereof. In some cases, thebiomarkers described herein comprise genes: TXNRD2, TXN2, MSRB3, MSRA,GSTZ1, NQO2, GSTT2, GSTM3, GLRX, GSTO1, GLRX3, TXNRD1, SELO, PON1, andSEPXI and the proteins encoded by the respective genes.

In some embodiments, the expression level of one or more biomarkersdescribed herein is determined at the nucleic acid level. Nucleicacid-based techniques for assessing expression are well known in the artand include, for example, determining the level of biomarker mRNA in abiological sample. Many expression detection methods use isolated RNA.Any RNA isolation technique that does not select against the isolationof mRNA is utilized for the purification of RNA (see, e.g., Ausubel etal., ed. (1987-1999) Current Protocols in Molecular Biology (John Wiley& Sons, New York). Additionally, large numbers of tissue samples arereadily processed using techniques well known to those of skill in theart, such as, for example, the single-step RNA isolation processdisclosed in U.S. Pat. No. 4,843,155.

As used herein, the term “nucleic acid probe” refers to any moleculethat is capable of selectively binding to a specifically intended targetnucleic acid molecule, for example, a nucleotide transcript. Suitablemethods for synthesizing nucleic acid probes are also described inCaruthers, Science, 230:281-285, (1985). In some instances, probessuitable for use herein include those formed from nucleic acids, such asRNA and/or DNA, nucleic acid analogs, locked nucleic acids, modifiednucleic acids, and chimeric probes of a mixed class including a nucleicacid with another organic component such as peptide nucleic acids. Insome cases, probes are single stranded. In other cases, probes aredouble stranded. Exemplary nucleotide analogs include phosphate estersof deoxyadenosine, deoxy cytidine, deoxyguanosine, deoxythymidine,adenosine, cytidine, guanosine, and uridine. Other examples ofnon-natural nucleotides include a xanthine or hypoxanthine;5-bromouracil, 2-aminopurine, deoxyinosine, or methylated cytosine, suchas 5-methylcytosine, and N4-methoxy deoxy cytosine. Also included arebases of polynucleotide mimetics, such as methylated nucleic acids,e.g., 2′-0-methRNA, peptide nucleic acids, modified peptide nucleicacids, and any other structural moiety that can act substantially like anucleotide or base, for example, by exhibiting base-complementarity withone or more bases that occur in DNA or RNA.

In some cases, a probe used for detection optionally includes adetectable label, such as a radiolabel, fluorescent label, or enzymaticlabel. See for example Lancaster et al., U.S. Pat. No. 5,869,717. Insome embodiments, the probe is fluorescently labeled. Fluorescentlylabeled nucleotides may be produced by various techniques, such as thosedescribed in Karnbara et al., Bio/Technol., 6:816-21, (1988); Smith etal., Nucl. Acid Res., 13:2399-2412, (1985); and Smith et al., Nature,321: 674-679, (1986). The fluorescent dye may be linked to thedeoxyribose by a linker arm that is easily cleaved by chemical orenzymatic means. There are numerous linkers and methods for attachinglabels to nucleotides, as shown in Oligonucleotides and Analogues: APractical Approach, IRL Press, Oxford, (1991); Zuckerman et al.,Polynucleotides Res., 15: 5305-5321, (1987); Sharma et al.,Polynucleotides Res., 19:3019, (1991); Giusti et al., PCR Methods andApplications, 2:223-227, (1993); Fung et al. (U.S. Pat. No. 4,757,141);Stabinsky (U.S. Pat. No. 4,739,044); Agrawal et al., TetrahedronLetters, 31: 1543-1546, (1990); Sproat et al., Polynucleotides Res.,15:4837, (1987); and Nelson et al., Polynucleotides Res., 17:7187-7194,(1989). Extensive guidance exists in the literature for derivatizingfluorophore and quencher molecules for covalent attachment via commonreactive groups that may be added to a nucleotide. Many linking moietiesand methods for attaching fluorophore moieties to nucleotides alsoexist, as described in Oligonucleotides and Analogues, supra; Guisti etal., supra; Agrawal et al, supra; and Sproat et al., supra.

In some cases, the detectable label attached to the probe is eitherdirectly or indirectly detectable. In some embodiments, the exact labelmay be selected based, at least in part, on the particular type ofdetection method used. Exemplary detection methods include radioactivedetection, optical absorbance detection, e.g., UV-visible absorbancedetection, optical emission detection, e.g., fluorescence;phosphorescence or chemiluminescence; Raman scattering. Preferred labelsinclude optically-detectable labels, such as fluorescent labels.Examples of fluorescent labels include, but are not limited to,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives: acridine, acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-l-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-anilino- l-naphthyl)maleimide; anthranilamide, BODIPY; alexa;fluorescien; conjugated multi-dyes; Brilliant Yellow; coumarin andderivatives; coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanine dyes;cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI),5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid; 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-l-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives; eosin, eosin isothiocyanate, erythrosin and derivatives;erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives; 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6- carboxyfluorescein, fluorescein,fluorescein isothiocyanate, QFITC, (XRTTC); fluorescamine; IR144;lR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron.TM. Brilliant Red 3B-A) rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid;terbium chelate derivatives; Atto dyes, Cy3; Cy5; Cy5.5; Cy7; IRD 700;IRD 800; La Jolta. Blue, phthalo cyanine: and naphthalo cyanine. Labelsother than fluorescent labels are contemplated by the invention,including other optically-detectable labels.

Detection of a bound probe may be measured using any of a variety oftechniques dependent upon the label used, such as those known to one ofskill in the art. Exemplary detection methods include radioactivedetection, optical absorbance detection, e.g., UV-visible absorbancedetection, optical emission detection, e.g., fluorescence orchemiluminescence. Devices capable of sensing fluorescence from a singlemolecule include scanning tunneling microscope (siM) and the atomicforce microscope (AFM). Hybridization patterns may also be scanned usinga CCD camera (e.g., Model TE/CCD512SF, Princeton Instruments, Trenton,N.J.) with suitable optics (Ploem, in Fluorescent and Luminescent Probesfor Biological Activity Mason, T. G. Ed., Academic Press, Landon, pp.1-11 (1993)), such as described in Yershov et al., Proc. Natl. Acad.Sci. 93:-,1.913 (1996), or may be imaged by TV monitoring. Forradioactive signals, a phosphorimager device can be used (Johnston etal., Electrophoresis, 13:566, 1990; Drmanac et al., Electrophoresis,13:566, 1992; 1993). Other commercial suppliers of imaging instrumentsinclude General Scanning Inc., (Watertown, Mass, on the World Wide Webat genscan.com), Genix Technologies (Waterloo, Ontario, Canada; on theWorld Wide Web at confocal.com), and Applied Precision Inc.

In certain embodiments, the target nucleic acid or nucleic acid ligandor both are quantified using methods known in the art. For example,isolated mRNA are used in hybridization or amplification assays thatinclude, but are not limited to, Southern or Northern analyses,polymerase chain reaction analyses and probe arrays. One method for thedetection of mRNA levels involves contacting the isolated mRNA with anucleic acid molecule (probe) that hybridize to the mRNA encoded by thegene being detected. The nucleic acid probe comprises of, for example, afull-length cDNA, or a portion thereof, such as an oligonucleotide of atleast 7, 15, 30, 50, 100, 250 or 500 nucleotides in length andsufficient to specifically hybridize under stringent conditions to anmRNA or genomic DNA encoding a biomarker, biomarker described hereinabove. Hybridization of an mRNA with the probe indicates that thebiomarker or other target protein of interest is being expressed.

In one embodiment, the mRNA is immobilized on a solid surface andcontacted with a probe, for example by running the isolated mRNA on anagarose gel and transferring the mRNA from the gel to a membrane, suchas nitrocellulose. In an alternative embodiment, the probe(s) areimmobilized on a solid surface and the mRNA is contacted with theprobe(s), for example, in a gene chip array. A skilled artisan readilyadapts known mRNA detection methods for use in detecting the level ofmRNA encoding the biomarkers or other proteins of interest.

An alternative method for determining the level of an mRNA of interestin a sample involves the process of nucleic acid amplification, e.g., byRT-PCR (see, for example, U.S. Pat. No. 4,683,202), ligase chainreaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189 193),self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl.Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwohet al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi et al. (1988) Bio/Technology 6:1197), rolling circlereplication (U.S. Pat. No. 5,854,033) or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers. In particular aspects of the invention, biomarker expression isassessed by quantitative fluorogenic RT-PCR (i.e., the TaqMan System).

Modifications or expression levels of an RNA of interest are monitoredusing a membrane blot (such as used in hybridization analysis such asNorthern, dot, and the like), or microwells, sample tubes, gels, beadsor fibers (or any solid support comprising bound nucleic acids). SeeU.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934,which are incorporated herein by reference. The detection of expressionalso comprises using nucleic acid probes in solution.

In some embodiments, microarrays are used to determine expression orpresence of one or more biomarkers. Microarrays are particularly wellsuited for this purpose because of the reproducibility between differentexperiments. DNA microarrays provide one method for the simultaneousmeasurement of the expression levels of large numbers of genes. Eacharray consists of a reproducible pattern of capture probes attached to asolid support. Labeled RNA or DNA is hybridized to complementary probeson the array and then detected by laser scanning Hybridizationintensities for each probe on the array are determined and converted toa quantitative value representing relative gene expression levels. See,U.S. Pat. Nos. 6,040,138, 5,800,992, 6,020,135, 6,033,860, 6,344,316,and U.S. Pat. Application 20120208706. High-density oligonucleotidearrays are particularly useful for determining the gene expressionprofile for a large number of RNA’s in a sample. Exemplary microarraychips include FoundationOne and FoundationOne Heme from FoundationMedicine, Inc; GeneChip® Human Genome U133 Plus 2.0 array fromAffymetrix; and Human DiscoveryMAP® 250+ v. 2.0 from Myraid RBM.

Techniques for the synthesis of these arrays using mechanical synthesismethods are described in, e.g., U.S. Pat. No. 5,384,261. In someembodiments, an array is fabricated on a surface of virtually any shapeor even a multiplicity of surfaces. In some embodiments, an array is aplanar array surface. In some embodiments, arrays include peptides ornucleic acids on beads, gels, polymeric surfaces, fibers such as fiberoptics, glass or any other appropriate substrate, see U.S. Pat. Nos.5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992, each of whichis hereby incorporated in its entirety for all purposes. In someembodiments, arrays are packaged in such a manner as to allow fordiagnostics or other manipulation of an all-inclusive device.

In some instances, a method for quantitation is quantitative polymerasechain reaction (QPCR). As used herein, “QPCR” refers to a PCR reactionperformed in such a way and under such controlled conditions that theresults of the assay are quantitative, that is, the assay is capable ofquantifying the amount or concentration of a nucleic acid ligand presentin the test sample. QPCR is a technique based on the polymerase chainreaction, and is used to amplify and simultaneously quantify a targetednucleic acid molecule. QPCR allows for both detection and quantification(as absolute number of copies or relative amount when normalized to DNAinput or additional normalizing genes) of a specific sequence in a DNAsample. The procedure follows the general principle of PCR, with theadditional feature that the amplified DNA is quantified as itaccumulates in the reaction in real time after each amplification cycle.QPCR is described, for example, in Kumit et al. (U.S. Pat. No.6,033,854), Wang et al. (U.S. Pat. Nos. 5,567,583 and 5,348,853), Ma etal. (The Journal of American Science, 2(3), (2006)), Heid et al. (GenomeResearch 986-994, (1996)), Sambrook and Russell (Quantitative PCR, ColdSpring Harbor Protocols, (2006)), and Higuchi (U.S. Pat. Nos. 6,171,785and 5,994,056).

In some embodiments, the expression level is a protein expression andthe level of the protein expression of a gene described herein isdetected. In some cases, the detection method comprises contacting abiological sample with an antibody that specifically recognizes orspecifically binds to a protein (e.g., a protein encoded by TXNRD2,TXN2, MSRB3, MSRA, GSTZ1, NQO2, GSTT2, GSTM3, GLRX, GSTO1, GLRX3,TXNRD1, SELO, PON1, or SEPX1) and detecting the complex between theantibody and the protein. In some cases, the antibody is an anti-TXNRD2antibody. In some cases, the antibody is an anti-TXN2 antibody. In someinstances, the antibody is an anti-MSRB3 antibody. In some cases, theantibody is an anti-MSRA antibody. In some cases, the antibody is ananti-GSTZ1 antibody. In some cases, the antibody is an anti-NQO2antibody. In some cases, the antibody is an anti-GSTT2 antibody. In somecases, the antibody is an anti-GSTM3 antibody. In some cases, theantibody is an anti-GLRX antibody. In some cases, the antibody is ananti-GSTO1 antibody. In some cases, the antibody is an anti-GLRX3antibody. In some cases, the antibody is an anti-TXNRD1 antibody. Insome cases, the antibody is an anti-SELO antibody. In some cases, theantibody is an anti-PON1 antibody. In some cases, the antibody is ananti-SEPX1 antibody. In some cases, the level of the protein expressionis determined by immunoassays including, but not limited to,radioimmunoassay, Western blot assay, ELISA, immunofluorescent assay,enzyme immunoassay, immunoprecipitation, chemiluminescent assay,immunohistochemical assay, dot blot assay, and slot blot assay.

In some instances, methylation analysis is carried out by any meansknown in the art. A variety of methylation analysis procedures are knownin the art and may be used to practice the methods disclosed herein.These assays allow for determination of the methylation state of one ora plurality of CpG sites within a tissue sample. In addition, thesemethods may be used for absolute or relative quantification ofmethylated nucleic acids. Such methylation assays involve, among othertechniques, two major steps. The first step is a methylation specificreaction or separation, such as (i) bisulfite treatment, (ii)methylation specific binding, or (iii) methylation specific restrictionenzymes. The second major step involves (i) amplification and detection,or (ii) direct detection, by a variety of methods such as (a) PCR(sequence-specific amplification) such as Taqman(R), (b) DNA sequencingof untreated and bisulfite-treated DNA, (c) sequencing by ligation ofdye-modified probes (including cyclic ligation and cleavage), (d)pyrosequencing, (e) single-molecule sequencing, (f) mass spectroscopy,or (g) Southern blot analysis.

Additionally, restriction enzyme digestion of PCR products amplifiedfrom bisulfite-converted DNA may be used, e.g., the method described bySadri and Hornsby (1996, Nucl. Acids Res. 24:5058- 5059), or COBRA(Combined Bisulfite Restriction Analysis) (Xiong and Laird, 1997,Nucleic Acids Res. 25:2532- 2534). COBRA analysis is a quantitativemethylation assay useful for determining DNA methylation levels atspecific gene loci in small amounts of genomic DNA. Briefly, restrictionenzyme digestion is used to reveal methylation-dependent sequencedifferences in PCR products of sodium bisulfite- treated DNA.Methylation-dependent sequence differences are first introduced into thegenomic DNA by standard bisulfite treatment according to the proceduredescribed by Frommer et al. (Frommer et al, 1992, Proc. Nat. Acad. Sci.USA, 89, 1827-1831). PCR amplification of the bisulfite converted DNA isthen performed using primers specific for the CpG sites of interest,followed by restriction endonuclease digestion, gel electrophoresis, anddetection using specific, labeled hybridization probes. Methylationlevels in the original DNA sample are represented by the relativeamounts of digested and undigested PCR product in a linearlyquantitative fashion across a wide spectrum of DNA methylation levels.In addition, this technique can be reliably applied to DNA obtained frommicro-dissected paraffin- embedded tissue samples. Typical reagents(e.g., as might be found in a typical COBRA- based kit) for COBRAanalysis may include, but are not limited to: PCR primers for specificgene (or methylation-altered DNA sequence or CpG island); restrictionenzyme and appropriate buffer; gene-hybridization oligo; controlhybridization oligo; kinase labeling kit for oligo probe; andradioactive nucleotides. Additionally, bisulfite conversion reagents mayinclude: DNA denaturation buffer; sulfo nation buffer; DNA recoveryreagents or kits (e.g., precipitation, ultrafiltration, affinitycolumn); desulfonation buffer; and DNA recovery components.

In an embodiment, the methylation profile of selected CpG sites isdetermined using methylation-Specific PCR (MSP). MSP allows forassessing the methylation status of virtually any group of CpG siteswithin a CpG island, independent of the use of methylation- sensitiverestriction enzymes (Herman et al, 1996, Proc. Nat. Acad. Sci. USA, 93,9821- 9826; U.S. Pat. Nos. 5,786,146, 6,017,704, 6,200,756, 6,265,171(Herman and Baylin); U.S. Pat. Pub. No. 2010/0144836 (Van Engeland etal); which are hereby incorporated by reference in their entirety).Briefly, DNA is modified by a deaminating agent such as sodium bisulfiteto convert unmethylated, but not methylated cytosines to uracil, andsubsequently amplified with primers specific for methylated versusunmethylated DNA. In some instances, typical reagents (e.g., as might befound in a typical MSP- based kit) for MSP analysis include, but are notlimited to: methylated and unmethylated PCR primers for specific gene(or methylation- altered DNA sequence or CpG island), optimized PCRbuffers and deoxynucleotides, and specific probes. One may usequantitative multiplexed methylation specific PCR (QM-PCR), as describedby Fackler et al. Fackler et al, 2004, Cancer Res. 64(13) 4442-4452; orFackler et al, 2006, Clin. Cancer Res. 12(11 Pt 1) 3306-3310.

In an embodiment, the methylation profile of selected CpG sites isdetermined using MethyLight and/or Heavy Methyl Methods. The MethyLightand Heavy Methyl assays are a high-throughput quantitative methylationassay that utilizes fluorescence- based real-time PCR (Taq Man(R))technology that requires no further manipulations after the PCR step(Eads, C.A. et al, 2000, Nucleic Acid Res. 28, e 32; Cottrell et al,2007, J. Urology 177, 1753, U.S. Pat. Nos. 6,331,393 (Laird et al), thecontents of which are hereby incorporated by reference in theirentirety). Briefly, the MethyLight process begins with a mixed sample ofgenomic DNA that is converted, in a sodium bisulfite reaction, to amixed pool of methylation-dependent sequence differences according tostandard procedures (the bisulfite process converts unmethylatedcytosine residues to uracil). Fluorescence-based PCR is then performedeither in an “unbiased” (with primers that do not overlap known CpGmethylation sites) PCR reaction, or in a “biased” (with PCR primers thatoverlap known CpG dinucleotides) reaction. In some cases, sequencediscrimination occurs either at the level of the amplification processor at the level of the fluorescence detection process, or both. In somecases, the MethyLight assay is used as a quantitative test formethylation patterns in the genomic DNA sample, wherein sequencediscrimination occurs at the level of probe hybridization. In thisquantitative version, the PCR reaction provides for unbiasedamplification in the presence of a fluorescent probe that overlaps aparticular putative methylation site. An unbiased control for the amountof input DNA is provided by a reaction in which neither the primers, northe probe overlie any CpG dinucleotides. Alternatively, a qualitativetest for genomic methylation is achieved by probing of the biased PCRpool with either control oligonucleotides that do not “cover” knownmethylation sites (a fluorescence- based version of the “MSP”technique), or with oligonucleotides covering potential methylationsites. Typical reagents (e.g., as might be found in a typicalMethyLight- based kit) for MethyLight analysis may include, but are notlimited to: PCR primers for specific gene (or methylation-altered DNAsequence or CpG island); TaqMan(R) probes; optimized PCR buffers anddeoxynucleotides; and Taq polymerase.

Quantitative MethyLight uses bisulfite to convert genomic DNA and themethylated sites are amplified using PCR with methylation independentprimers. Detection probes specific for the methylated and unmethylatedsites with two different fluorophores provides simultaneous quantitativemeasurement of the methylation. The Heavy Methyl technique begins withbisulfate conversion of DNA. Next specific blockers prevent theamplification of unmethylated DNA. Methylated genomic DNA does not bindthe blockers and their sequences will be amplified. The amplifiedsequences are detected with a methylation specific probe. (Cottrell etal, 2004, Nuc. Acids Res. 32:e10, the contents of which is herebyincorporated by reference in its entirety).

The Ms-SNuPE technique is a quantitative method for assessingmethylation differences at specific CpG sites based on bisulfitetreatment of DNA, followed by single-nucleotide primer extension(Gonzalgo and Jones, 1997, Nucleic Acids Res. 25, 2529-2531). Briefly,genomic DNA is reacted with sodium bisulfite to convert unmethylatedcytosine to uracil while leaving 5-methylcytosine unchanged.Amplification of the desired target sequence is then performed using PCRprimers specific for bisulfite-converted DNA, and the resulting productis isolated and used as a template for methylation analysis at the CpGsite(s) of interest. In some cases, small amounts of DNA are analyzed(e.g., micro-dissected pathology sections), and the method avoidsutilization of restriction enzymes for determining the methylationstatus at CpG sites. Typical reagents (e.g., as is found in a typicalMs-SNuPE-based kit) for Ms-SNuPE analysis include, but are not limitedto: PCR primers for specific gene (or methylation-altered DNA sequenceor CpG island); optimized PCR buffers and deoxynucleotides; gelextraction kit; positive control primers; Ms-SNuPE primers for specificgene; reaction buffer (for the Ms-SNuPE reaction); and radioactivenucleotides. Additionally, bisulfite conversion reagents may include:DNA denaturation buffer; sulfonation buffer; DNA recovery regents or kit(e.g., precipitation, ultrafiltration, affinity column); desulfonationbuffer; and DNA recovery components.

In another embodiment, the methylation status of selected CpG sites isdetermined using differential Binding-based Methylation DetectionMethods. For identification of differentially methylated regions, oneapproach is to capture methylated DNA. This approach uses a protein, inwhich the methyl binding domain of MBD2 is fused to the Fc fragment ofan antibody (MBD-FC) (Gebhard et al, 2006, Cancer Res. 66:6118-6128; andPCT Pub. No. WO 2006/056480 A2 (Relhi), the contents of which are herebyincorporated by reference in their entirety). This fusion protein hasseveral advantages over conventional methylation specific antibodies.The MBD FC has a higher affinity to methylated DNA and it binds doublestranded DNA. Most importantly the two proteins differ in the way theybind DNA. Methylation specific antibodies bind DNA stochastically, whichmeans that only a binary answer can be obtained. The methyl bindingdomain of MBD-FC, on the other hand, binds DNA molecules regardless oftheir methylation status. The strength of this protein - DNA interactionis defined by the level of DNA methylation. After binding genomic DNA,eluate solutions of increasing salt concentrations can be used tofractionate non- methylated and methylated DNA allowing for a morecontrolled separation (Gebhard et al, 2006, Nucleic Acids Res. 34: e82).Consequently this method, called Methyl-CpG immunoprecipitation (MCIP),not only enriches, but also fractionates genomic DNA according tomethylation level, which is particularly helpful when the unmethylatedDNA fraction should be investigated as well.

In an alternative embodiment, a 5 -methyl cytidine antibody to bind andprecipitate methylated DNA. Antibodies are available from Abeam(Cambridge, MA), Diagenode (Sparta, NJ) or Eurogentec (c/o AnaSpec,Fremont, CA). Once the methylated fragments have been separated they maybe sequenced using microarray based techniques such as methylatedCpG-island recovery assay (MIRA) or methylated DNA immunoprecipitation(MeDIP) (Pelizzola et al, 2008, Genome Res. 18, 1652-1659; O’Geen et al,2006, BioTechniques 41(5), 577-580, Weber et al, 2005, Nat. Genet. 37,853-862; Horak and Snyder, 2002, Methods Enzymol, 350, 469-83; Lieb,2003, Methods Mol Biol, 224, 99-109). Another technique is methyl-CpGbinding domain column/segregation of partly melted molecules (MBD/SPM,Shiraishi et al, 1999, Proc. Natl. Acad. Sci. USA 96(6):2913-2918).

In some embodiments, methods for detecting methylation include randomlyshearing or randomly fragmenting the genomic DNA, cutting the DNA with amethylation-dependent or methylation-sensitive restriction enzyme andsubsequently selectively identifying and/or analyzing the cut or uncutDNA. Selective identification can include, for example, separating cutand uncut DNA (e.g., by size) and quantifying a sequence of interestthat was cut or, alternatively, that was not cut. See, e.g., U.S. Pat.No. 7,186,512. Alternatively, the method can encompass amplifying intactDNA after restriction enzyme digestion, thereby only amplifying DNA thatwas not cleaved by the restriction enzyme in the area amplified. See,e.g., U.S. Pats. No. 7,910,296; No. 7,901,880; and No. 7,459,274. Insome embodiments, amplification can be performed using primers that aregene specific.

For example, there are methyl-sensitive enzymes that preferentially orsubstantially cleave or digest at their DNA recognition sequence if itis non-methylated. Thus, an unmethylated DNA sample is cut into smallerfragments than a methylated DNA sample. Similarly, a hypermethylated DNAsample is not cleaved. In contrast, there are methyl-sensitive enzymesthat cleave at their DNA recognition sequence only if it is methylated.Methyl- sensitive enzymes that digest unmethylated DNA suitable for usein methods of the technology include, but are not limited to, Hpall,Hhal, Maell, BstUI and Acil. In some instances, an enzyme that is usedis Hpall that cuts only the unmethylated sequence CCGG. In otherinstances, another enzyme that is used is Hhal that cuts only theunmethylated sequence GCGC. Both enzymes are available from New EnglandBioLabs(R), Inc. Combinations of two or more methyl-sensitive enzymesthat digest only unmethylated DNA are also used. Suitable enzymes thatdigest only methylated DNA include, but are not limited to, Dpnl, whichonly cuts at fully methylated 5′-GATC sequences, and McrBC, anendonuclease, which cuts DNA containing modified cytosines(5-methylcytosine or 5-hydroxymethylcytosine or N4-methylcytosine) andcuts at recognition site 5′... PumC(N4o-3ooo) PumC... 3′ (New EnglandBioLabs, Inc., Beverly, MA). Cleavage methods and procedures forselected restriction enzymes for cutting DNA at specific sites are wellknown to the skilled artisan. For example, many suppliers of restrictionenzymes provide information on conditions and types of DNA sequences cutby specific restriction enzymes, including New England BioLabs, Pro-MegaBiochems, Boehringer-Mannheim, and the like. Sambrook et al. (SeeSambrook et al. Molecular Biology: A Laboratory Approach, Cold SpringHarbor, N.Y. 1989) provide a general description of methods for usingrestriction enzymes and other enzymes.

In some instances, a methylation-dependent restriction enzyme is arestriction enzyme that cleaves or digests DNA at or in proximity to amethylated recognition sequence, but does not cleave DNA at or near thesame sequence when the recognition sequence is not methylated.Methylation-dependent restriction enzymes include those that cut at amethylated recognition sequence (e.g., Dpnl) and enzymes that cut at asequence near but not at the recognition sequence (e.g., McrBC). Forexample, McrBC’s recognition sequence is 5′ RmC (N40-3000) RmC 3 ′ where“R” is a purine and “mC” is a methylated cytosine and “N40-3000”indicates the distance between the two RmC half sites for which arestriction event has been observed. McrBC generally cuts close to onehalf-site or the other, but cleavage positions are typically distributedover several base pairs, approximately 30 base pairs from the methylatedbase. McrBC sometimes cuts 3′ of both half sites, sometimes 5′ of bothhalf sites, and sometimes between the two sites. Exemplarymethylation-dependent restriction enzymes include, e.g., McrBC, McrA,MrrA, Bisl, Glal and Dpnl. One of skill in the art will appreciate thatany methylation-dependent restriction enzyme, including homologs andorthologs of the restriction enzymes described herein, is also suitablefor use with one or more methods described herein.

In some cases, a methylation-sensitive restriction enzyme is arestriction enzyme that cleaves DNA at or in proximity to anunmethylated recognition sequence but does not cleave at or in proximityto the same sequence when the recognition sequence is methylated.Exemplary methylation-sensitive restriction enzymes are described in,e.g., McClelland et al, 22(17) NUCLEIC ACIDS RES. 3640-59 (1994).Suitable methylation-sensitive restriction enzymes that do not cleaveDNA at or near their recognition sequence when a cytosine within therecognition sequence is methylated at position C5 include, e.g., Aat II,Aci I, Acd I, Age I, Alu I, Asc I, Ase I, AsiS I, Bbe I, BsaA I, BsaH I,BsiE I, BsiW I, BsrF I, BssH II, BssK I, BstB I, BstN I, BstU I, Cla I,Eae I, Eag I, Fau I, Fse I, Hha I, HinPl I, HinC II, Hpa II, Hpy99 I,HpyCH4 IV, Kas I, Mbo I, Mlu I, MapAl I, Msp I, Nae I, Nar I, Not I, PmlI, Pst I, Pvu I, Rsr II, Sac II, Sap I, Sau3A I, Sfl I, Sfo I, SgrA I,Sma I, SnaB I, Tsc I, Xma I, and Zra I. Suitable methylation-sensitiverestriction enzymes that do not cleave DNA at or near their recognitionsequence when an adenosine within the recognition sequence is methylatedat position N6 include, e.g., Mbo I. One of skill in the art willappreciate that any methylation-sensitive restriction enzyme, includinghomologs and orthologs of the restriction enzymes described herein, isalso suitable for use with one or more of the methods described herein.One of skill in the art will further appreciate that amethylation-sensitive restriction enzyme that fails to cut in thepresence of methylation of a cytosine at or near its recognitionsequence may be insensitive to the presence of methylation of anadenosine at or near its recognition sequence. Likewise, amethylation-sensitive restriction enzyme that fails to cut in thepresence of methylation of an adenosine at or near its recognitionsequence may be insensitive to the presence of methylation of a cytosineat or near its recognition sequence. For example, Sau3AI is sensitive(i.e., fails to cut) to the presence of a methylated cytosine at or nearits recognition sequence, but is insensitive (i.e., cuts) to thepresence of a methylated adenosine at or near its recognition sequence.One of skill in the art will also appreciate that somemethylation-sensitive restriction enzymes are blocked by methylation ofbases on one or both strands of DNA encompassing of their recognitionsequence, while other methylation-sensitive restriction enzymes areblocked only by methylation on both strands, but can cut if arecognition site is hemi-methylated.

In alternative embodiments, adaptors are optionally added to the ends ofthe randomly fragmented DNA, the DNA is then digested with amethylation-dependent or methylation-sensitive restriction enzyme, andintact DNA is subsequently amplified using primers that hybridize to theadaptor sequences. In this case, a second step is performed to determinethe presence, absence or quantity of a particular gene in an amplifiedpool of DNA. In some embodiments, the DNA is amplified using real-time,quantitative PCR.

In other embodiments, the methods comprise quantifying the averagemethylation density in a target sequence within a population of genomicDNA. In some embodiments, the method comprises contacting genomic DNAwith a methylation-dependent restriction enzyme or methylation-sensitiverestriction enzyme under conditions that allow for at least some copiesof potential restriction enzyme cleavage sites in the locus to remainuncleaved; quantifying intact copies of the locus; and comparing thequantity of amplified product to a control value representing thequantity of methylation of control DNA, thereby quantifying the averagemethylation density in the locus compared to the methylation density ofthe control DNA.

The methylated CpG island amplification (MCA) technique is a method thatcan be used to screen for altered methylation patterns in genomic DNA,and to isolate specific sequences associated with these changes (Toyotaet al, 1999, Cancer Res. 59, 2307-2312, U.S. Pat. No. 7,700,324 (Issa etal), the contents of which are hereby incorporated by reference in theirentirety). Briefly, restriction enzymes with different sensitivities tocytosine methylation in their recognition sites are used to digestgenomic DNAs from primary tumors, cell lines, and normal tissues priorto arbitrarily primed PCR amplification. Fragments that showdifferential methylation are cloned and sequenced after resolving thePCR products on high-resolution polyacrylamide gels. The clonedfragments are then used as probes for Southern analysis to confirmdifferential methylation of these regions. Typical reagents (e.g., asmight be found in a typical MCA-based kit) for MCA analysis may include,but are not limited to: PCR primers for arbitrary priming Genomic DNA;PCR buffers and nucleotides, restriction enzymes and appropriatebuffers; gene-hybridization oligos or probes; control hybridizationoligos or probes.

Additional methylation detection methods include those methods describedin, e.g., U.S. Pats. No. 7,553,627; No. 6,331,393; U.S. Pat. Serial No.12/476,981; U.S. Pat. Publication No. 2005/0069879; Rein, et al, 26(10)NUCLEIC ACIDS RES. 2255-64 (1998); and Olek et al, 17(3) NAT. GENET.275-6 (1997).

In another embodiment, the methylation status of selected CpG sites isdetermined using Methylation- Sensitive High Resolution Melting (HRM).Recently, Wojdacz et al. reported methylation-sensitive high resolutionmelting as a technique to assess methylation. (Wojdacz and Dobrovic,2007, Nuc. Acids Res. 35(6) e41; Wojdacz et al. 2008, Nat. Prot. 3(12)1903-1908; Balic et al, 2009 J. Mol. Diagn. 11 102- 108; and U.S. Pat.Pub. No. 2009/0155791 (Wojdacz et al), the contents of which are herebyincorporated by reference in their entirety). A variety of commerciallyavailable real time PCR machines have HRM systems including the RocheLightCycler480, Corbett Research RotorGene6000, and the AppliedBiosystems 7500. HRM may also be combined with other amplificationtechniques such as pyrosequencing as described by Candiloro et al.(Candiloro et al, 2011, Epigenetics 6(4) 500-507).

In another embodiment, the methylation status of selected CpG locus isdetermined using a primer extension assay, including an optimized PCRamplification reaction that produces amplified targets for analysisusing mass spectrometry. The assay can also be done in multiplex. Massspectrometry is a particularly effective method for the detection ofpolynucleotides associated with the differentially methylated regulatoryelements. The presence of the polynucleotide sequence is verified bycomparing the mass of the detected signal with the expected mass of thepolynucleotide of interest. The relative signal strength, e.g., masspeak on a spectra, for a particular polynucleotide sequence indicatesthe relative population of a specific allele, thus enabling calculationof the allele ratio directly from the data. This method is described indetail in PCT Pub. No. WO 2005/012578A1 (Beaulieu et al), which ishereby incorporated by reference in its entirety. For methylationanalysis, the assay can be adopted to detect bisulfite introducedmethylation dependent C to T sequence changes. These methods areparticularly useful for performing multiplexed amplification reactionsand multiplexed primer extension reactions (e.g., multiplexedhomogeneous primer mass extension (hME) assays) in a single well tofurther increase the throughput and reduce the cost per reaction forprimer extension reactions.

Other methods for DNA methylation analysis include restriction landmarkgenomic scanning (RLGS, Costello et al, 2002, Meth. Mol Biol, 200,53-70), methylation- sensitive-representational difference analysis(MS-RDA, Ushijima and Yamashita, 2009, Methods Mol Biol 507, 1 17-130).Comprehensive high-throughput arrays for relative methylation (CHARM)techniques are described in WO 2009/021141 (Feinberg and Irizarry). TheRoche(R) NimbleGen(R) microarrays including the ChromatinImmunoprecipitation-on- chip (ChlP-chip) or methylated DNAimmunoprecipitation-on-chip (MeDIP-chip). These tools have been used fora variety of cancer applications including melanoma, liver cancer andlung cancer (Koga et al, 2009, Genome Res., 19, 1462-1470; Acevedo etal, 2008, Cancer Res., 68, 2641-2651; Rauch et al, 2008, Proc. Nat.Acad. Sci. USA, 105, 252-257). Others have reported bisulfateconversion, padlock probe hybridization, circularization, amplificationand next generation or multiplexed sequencing for high throughputdetection of methylation (Deng et al, 2009, Nat. Biotechnol 27, 353-360;Ball et al, 2009, Nat. Biotechnol 27, 361-368; U.S. Pat. No. 7,611,869(Fan)). As an alternative to bisulfate oxidation, Bayeyt et al. havereported selective oxidants that oxidize 5-methylcytosine, withoutreacting with thymidine, which are followed by PCR or pyro sequencing(WO 2009/049916 (Bayeyt et al).

Pharmaceutical Formulations, Dosage Forms and Treatment Regimens

Another aspect of the present invention relates to formulations androutes of administration for pharmaceutical compositions comprising anitrobenzamide compound. Such pharmaceutical compositions can be used totreat cancer in the methods described in detail above.

The compounds of Formula I may be provided as a prodrug and/or may beallowed to interconvert to a nitrosobenzamide form in vivo afteradministration. That is, either the nitrobenzamide form and/or thenitrosobenzamide form, or pharmaceutically acceptable salts may be usedin developing a formulation for use in the present invention. Further,in some embodiments, the compound may be used in combination with one ormore other compounds or in one or more other forms. For example aformulation may comprise both the nitrobenzamide compound and acid formsin particular proportions, depending on the relative potencies of eachand the intended indication. The two forms may be formulated together,in the same dosage unit e.g. in one cream, suppository, tablet, capsule,or packet of powder to be dissolved in a beverage; or each form may beformulated in a separate unit, e.g., two creams, two suppositories, twotablets, two capsules, a tablet and a liquid for dissolving the tablet,a packet of powder and a liquid for dissolving the powder, etc.

In compositions comprising combinations of a nitrobenzamide compound andanother active agent can be effective. The two compounds and/or forms ofa compound may be formulated together, in the same dosage unit e.g. inone cream, suppository, tablet, capsule, or packet of powder to bedissolved in a beverage; or each form may be formulated in separateunits, e.g., two creams, suppositories, tablets, two capsules, a tabletand a liquid for dissolving the tablet, a packet of powder and a liquidfor dissolving the powder, etc.

The term “pharmaceutically acceptable salt” means those salts whichretain the biological effectiveness and properties of the compounds usedin the present invention, and which are not biologically or otherwiseundesirable. For example, a pharmaceutically acceptable salt does notinterfere with the beneficial effect of the compound of the invention intreating a cancer.

Typical salts are those of the inorganic ions, such as, for example,sodium, potassium, calcium and magnesium ions. Such salts include saltswith inorganic or organic acids, such as hydrochloric acid, hydrobromicacid, phosphoric acid, nitric acid, sulfuric acid, methanesulfonic acid,p-toluenesulfonic acid, acetic acid, fumaric acid, succinic acid, lacticacid, mandelic acid, malic acid, citric acid, tartaric acid or maleicacid. In addition, if the compounds used in the present inventioncontain a carboxy group or other acidic group, it may be converted intoa pharmaceutically acceptable addition salt with inorganic or organicbases. Examples of suitable bases include sodium hydroxide, potassiumhydroxide, ammonia, cyclohexylamine, dicyclohexyl-amine, ethanolamine,diethanolamine and triethanolamine.

For oral administration, the compounds can be formulated readily bycombining the active compound(s) with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, including chewable tablets,pills, dragees, capsules, lozenges, hard candy, liquids, gels, syrups,slurries, powders, suspensions, elixirs, wafers, and the like, for oralingestion by a patient to be treated. Such formulations can comprisepharmaceutically acceptable carriers including solid diluents orfillers, sterile aqueous media and various non-toxic organic solvents.Generally, the compounds of the invention will be included atconcentration levels ranging from about 0.5%, about 5%, about 10%, about20%, or about 30% to about 50%, about 60%, about 70%, about 80% or about90% by weight of the total composition of oral dosage forms, in anamount sufficient to provide a desired unit of dosage.

Aqueous suspensions may contain a nitrobenzamide compound withpharmaceutically acceptable excipients, such as a suspending agent(e.g., methyl cellulose), a wetting agent (e.g., lecithin, lysolecithinand/or a long-chain fatty alcohol), as well as coloring agents,preservatives, flavoring agents, and the like.

In some embodiments, oils or non-aqueous solvents may be required tobring the compounds into solution, due to, for example, the presence oflarge lipophilic moieties. Alternatively, emulsions, suspensions, orother preparations, for example, liposomal preparations, may be used.With respect to liposomal preparations, any known methods for preparingliposomes for treatment of a condition may be used. See, for example,Bangham et al., J. Mol. Biol, 23: 238-252 (1965) and Szoka et al., Proc.Natl Acad. Sci 75: 4194-4198 (1978), incorporated herein by reference.Ligands may also be attached to the liposomes to direct thesecompositions to particular sites of action. Compounds of this inventionmay also be integrated into foodstuffs, e.g, cream cheese, butter, saladdressing, or ice cream to facilitate solubilization, administration,and/or compliance in certain patient populations.

Pharmaceutical preparations for oral use may be obtained as a solidexcipient, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; flavoring elements, cellulose preparations such as, forexample, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinyl pyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. The compounds may alsobe formulated as a sustained release preparation.

Dragee cores can be provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for administration.

For injection, the inhibitors of the present invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank’s solution, Ringer’s solution, or physiological salinebuffer. Such compositions may also include one or more excipients, forexample, preservatives, solubilizers, fillers, lubricants, stabilizers,albumin, and the like. Methods of formulation are known in the art, forexample, as disclosed in Remington’s Pharmaceutical Sciences, latestedition, Mack Publishing Co., Easton Pa. These compounds may also beformulated for transmucosal administration, buccal administration, foradministration by inhalation, for parental administration, fortransdermal administration, and rectal administration.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation or transcutaneous delivery (forexample subcutaneously or intramuscularly), intramuscular inj ection oruse of a transdermal patch. Thus, for example, the compounds may beformulated with suitable polymeric or hydrophobic materials (for exampleas an emulsion in an acceptable oil) or ion exchange resins, or assparingly soluble derivatives, for example, as a sparingly soluble salt.

As described elsewhere herein, in some instances 4-iodo-3-nitrobenzamideor a salt, metabolite or prodrug thereof is administered from about 5mg/kg to about 200 mg/kg, from about 5 mg/kg to about 150 mg/kg, fromabout 5 mg/kg to about 100 mg/kg, or from about 5 mg/kg to about 60mg/kg. In other instances, 4-iodo-3-nitrobenzamide or a salt, metaboliteor prodrug thereof is administered at about 5 mg/kg, about 6 mg/kg,about 7 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 8.6 mg/kg, about 9mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg,about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, or about 60 mg/kg.

In the case wherein the patient’s status does improve, upon the doctor’sdiscretion the administration of the compounds may be givencontinuously; alternatively, the dose of drug being administered may betemporarily reduced or temporarily suspended for a certain length oftime (i.e., a “drug holiday”). The length of the drug holiday can varybetween 2 days and 1 year, including by way of example only, 2 days, 3days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days,180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or365 days. The dose reduction during a drug holiday may be from 10%-100%,including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Once improvement of the patient’s conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, can be reduced, as a function ofthe symptoms, to a level at which the improved disease, disorder orcondition is retained. Patients can, however, require intermittenttreatment on a long-term basis upon any recurrence of symptoms.

The amount of a given agent that will correspond to such an amount willvary depending upon factors such as the particular compound, theseverity of the disease, the identity (e.g., weight) of the subject orhost in need of treatment, but can nevertheless be routinely determinedin a manner known in the art according to the particular circumstancessurrounding the case, including, e.g., the specific agent beingadministered, the route of administration, and the subject or host beingtreated. The desired dose may conveniently be presented in a single doseor as divided doses administered simultaneously (or over a short periodof time) or at appropriate intervals, for example as two, three, four ormore sub-doses per day.

The foregoing ranges are merely suggestive, as the number of variablesin regard to an individual treatment regime is large, and considerableexcursions from these recommended values are not uncommon. Such dosagesmay be altered depending on a number of variables, not limited to theactivity of the compound used, the disease or condition to be treated,the mode of administration, the requirements of the individual subject,the severity of the disease or condition being treated, and the judgmentof the practitioner.

Toxicity and therapeutic efficacy of such therapeutic regimens can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, including, but not limited to, the determinationof the LD50 (the dose lethal to 50% of the population) and the ED50 (thedose therapeutically effective in 50% of the population). The dose ratiobetween the toxic and therapeutic effects is the therapeutic index andit can be expressed as the ratio between LD50 and ED50. Compoundsexhibiting high therapeutic indices are preferred. The data obtainedfrom cell culture assays and animal studies can be used in formulating arange of dosage for use in human. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED50 with minimal toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized.

Kits/Article of Manufacture

Disclosed herein, in certain embodiments, are kits and articles ofmanufacture for use with one or more methods described herein. Such kitsinclude a carrier, package, or container that is compartmentalized toreceive one or more containers such as vials, tubes, and the like, eachof the container(s) comprising one of the separate elements to be usedin a method described herein. Suitable containers include, for example,bottles, vials, syringes, and test tubes. In one embodiment, thecontainers are formed from a variety of materials such as glass orplastic.

The articles of manufacture provided herein contain packaging materials.Examples of pharmaceutical packaging materials include, but are notlimited to, blister packs, bottles, tubes, bags, containers, bottles,and any packaging material suitable for a selected formulation andintended mode of administration and treatment.

For example, the container(s) include iniparib, optionally in acomposition or in combination with temozolomide. Such kits optionallyinclude an identifying description or label or instructions relating toits use in the methods described herein.

A kit typically includes labels listing contents and/or instructions foruse, and package inserts with instructions for use. A set ofinstructions will also typically be included.

In one embodiment, a label is on or associated with the container. Inone embodiment, a label is on a container when letters, numbers or othercharacters forming the label are attached, molded or etched into thecontainer itself; a label is associated with a container when it ispresent within a receptacle or carrier that also holds the container,e.g., as a package insert. In one embodiment, a label is used toindicate that the contents are to be used for a specific therapeuticapplication. The label also indicates directions for use of thecontents, such as in the methods described herein.

In certain embodiments, the pharmaceutical compositions are presented ina pack or dispenser device which contains one or more unit dosage formscontaining a compound provided herein. The pack, for example, containsmetal or plastic foil, such as a blister pack. In one embodiment, thepack or dispenser device is accompanied by instructions foradministration. In one embodiment, the pack or dispenser is alsoaccompanied with a notice associated with the container in formprescribed by a governmental agency regulating the manufacture, use, orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the drug for human or veterinary administration.Such notice, for example, is the labeling approved by the U.S. Food andDrug Administration for prescription drugs, or the approved productinsert. In one embodiment, compositions containing a compound providedherein formulated in a compatible pharmaceutical carrier are alsoprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

Certain Terminology

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the claimed subject matter belongs. It is to be understoodthat the foregoing general description and the following detaileddescription are exemplary and explanatory only and are not restrictiveof any subject matter claimed. In this application, the use of thesingular includes the plural unless specifically stated otherwise. Itmust be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. In this application, theuse of “or” means “and/or” unless stated otherwise. Furthermore, use ofthe term “including” as well as other forms, such as “include”,“includes,” and “included,” is not limiting.

As used herein, ranges and amounts can be expressed as “about” aparticular value or range. About also includes the exact amount. Hence“about 5 µL” means “about 5 µL” and also “5 µL.” Generally, the term“about” includes an amount that would be expected to be withinexperimental error.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

As used herein, the terms “individual(s)”, “subject(s)” and “patient(s)”mean any mammal. In some embodiments, the mammal is a human. In someembodiments, the mammal is a non-human. None of the terms require or arelimited to situations characterized by the supervision (e.g. constant orintermittent) of a health care worker (e.g. a doctor, a registerednurse, a nurse practitioner, a physician’s assistant, an orderly or ahospice worker).

As used herein, the term “first-line treatment” refers to a primarytreatment for a subject with a cancer. In some instances, the cancer isa primary cancer. In other instances, the cancer is a metastatic orrecurrent cancer. In some cases, the first-line treatment compriseschemotherapy. In other cases, the first-line treatment comprisesradiation therapy. A skilled artisan would readily understand thatdifferent first-line treatments may be applicable to different type ofcancers.

EXAMPLES

These examples are provided for illustrative purposes only and not tolimit the scope of the claims provided herein.

Example 1 - Phase I Clinical Trial - Glioblastoma Multiforme (GBM)

The Iniparib phase 1 GBM study was a multicenter study. It was a singlearm, multi dose, dose escalating trial in newly diagnosed GBM patients.The total number of patients was 43, with about 5 patients per cohort.Patients who tolerated radiation (XRT) and temozolomide (TMZ) wererecruited. Inclusion criteria included completion of XRT and TMZ withoutgrade 3 or 4 toxicity and labs within acceptable range within 6 weeks ofcompleting XRT. The end points included safety, maximum tolerated dose(MTD), and signal of activity.

The patients were separated into two study groups with the followingtreatment schema (also see FIG. 1 ).

Study Group 1 Treatment Cycles 4 Weeks Each (N=23)

Cycle 1

-   Days 1-5: TMZ 150 mg/m²-   Weeks 1-4: BSI-201, starting dose 5.1 mg/kg

Cycle 2

-   Days 1-5: TMZ 200 mg/m²-   Weeks 1-4: BSI-201, starting dose 5.1 mg/kg-   MRI performed after every odd cycle (every 8 weeks) until    progression.

Study Group 2 Treatment Cycles 10 Weeks Each (N=20)

-   Weeks 1-6: TMZ Daily 75 mg/m²-   Weeks 1-6: BSI-201, starting dose 5.1 mg/kg Weeks 7-10: Rest, no    treatment-   MRI performed after every cycle (every 10 weeks) until progression.

Continuous Reassessment Method (CRM) was used to determine doseescalation. PK was drawn at cycle 1, 2, 3 and off treatment. PD viaPBMCs was drawn at cycle 1, 2, 3 and off treatment. No cytochromeP450-inducing anticonvulsants. However, Gliadel was permitted.

In adjuvant phase:

-   Group 1: TMZ (150-200 mg/m² given 5 days/month x 6 cycles) -    standard dose with BSI-201 starting at 5.1 mg/kg.-   Group 2: TMZ (75 mg/m² daily 42 days on 30 days off x 3 cycles) -    metronomic dose with BSI-201 starting at 5.1 mg/kg.

Using modified continual reassessment method, MTD was defined formetronomic and standard dose TMZ. 6 dose levels were tested (lowest 5.1mg/kg - highest 9.5 mg/kg IV 2x/wk). At 8.6 mg/kg (17.2 mg/kg/week), ⅑patients had a DLT. The DLTs across both groups were: rash (1),hypersensitivity reaction (1), fatigue (1) and a thromboembolic event(1). Additional grade 3 toxicities were neutropenia, lymphopenia,nausea, and elevated AST. Phase 2 dose defined as 8 mg/kg IV 2x/wk withstandard TMZ and 8.6 mg/kg IV 2x/wk with metronomic TMZ.

Table 1 illustrates the pharmacokinetics of iniparib. Data are presentedas the geometric mean ± SD for peak plasma concentrations (C_(max)) andthe arithmetic average ± SD for the metabolite/iniparib concentrationratio as expressed as a percentage. Dosing is IV 2x/week continuous.IABM and IABA are the two major metabolites of iniparib in plasma.

Dose No. of No. of Cmax (ng/mL) Metabolite/iniparib (%) (mg/kg) Patientssamples Iniparib IABM IABA IABM IABA 5.1 8 20 642 ± 315 5.7 ± 4.1 16.6 ±5.2 1.1 ± 0.6 3.0 ± 2.0 6.1 5 11 928 ± 397 7.2 ± 1.8 14.9 ± 8.3 0.9 ±0.6 2.0 ± 1.5 6.8 7 18 1,019 ± 463 8.4 ± 3.7 19.9 ± 6.6 1.1 ± 0.9 2.4 ±1.4 8.0 4 5 3,687 ± 1,464 10.3 ± 2.9 14.5 ± 15.0 0.3 ± 0.1 0.7 ± 0.7 8.65 18 1,517 ± 987 9.7 ± 2.4 31.5 ± 10.4 0.8 ± 0.6 2.5 ± 1.4 9.5 4 7 2,139± 2,133 13.6 ± 5.3 19.8 ± 14.0 0.8 ± 0.6 2.0 ± 2.0

Table 2 illustrates the toxicity of iniparib. Grade 3-4 adverse eventsare shown with relationship of possible, or probable, or definite toiniparib.

Adverse Event Group I N=23 No. (% of pts) Group II N=20 No. (% of pts)Total N=43 No. (% of pts) Allergic reaction 1 (4) 1 (2) Alanine 6 (30) 6(14) Anemia 13 (57) 11 (55) 24 (56) Constipation 9 (39) 7 (35) 16 (37)Dizziness 5 (22) 5 (12) Fatigue 15 (65) 13 (65) 28 (65) Nausea 9 (39) 9(45) 18 (42) Rash maculo-papular 5 (22) 5 (12) Thromboembolic event 1(5) 1 (2) Lymphocyte count decreased 6 (30) 6 (14) Platelet decreased 15(65) 6 (30) 21 (49) White counts decreased 11 (48) 11 (55) 22 (51)

Example 2 - Phase II Clinical Trial - Glioblastoma Multiforme (GBM)

The primary objective of the Phase II study was to estimate the overallsurvival for adult patients with newly diagnosed glioblastoma multiforme(GBM) treated with BSI-201 (iniparib) at the MTDs during RT withconcurrent and adjuvant TMZ. The secondary objective was to estimate thefrequency of toxicity associated with this treatment regimen. 76patients were recruited for this study. Corollary studies includedPARP-1 expression in resected GBM and MGMT status in resected GBM.

Safety Run-In:

-   BSI-201 at one dose less than the MTD from Group 2 with TMX 75    mg/m² + XRT (3 patients), then-   BSI-201 at Group 2 MTD with TMX 75 mg/m² + XRT (3 patients) to    ensure safety of triple therapy.

The following treatment schema (also see FIG. 2 ).

Concomitant (6 weeks)

-   RT: 60 Gy (total) TMZ: Daily 75 mg/m²-   BSI-201: once per day, twice a week (8.0 mg/kg IV q2wk)

Rest (4 weeks) with no treatment. Maintenance Cycles 1-6 (4 weeks)

-   BSI-201: once per day, twice a week (8.6 mg/kg IV q2wk)-   TMZ: Days 1-5 (150-200 mg/m²), repeated every 28 days

For assessing the efficacy of the treatment in terms of overallsurvival, the overall failure rate were estimated and compared to thefailure rate of 0.6 per-person year of follow-up regarding the Phase IIItrial done by Stupp et al. in the same patient population treated withRT plus concomitant and adjuvant temozolomide.

The primary endpoint was death due to all causes. The survival time isdefined from time of histological diagnosis to death occurrence. Theoverall failure rate was expressed as hazard of failure per person-yearof follow-up. The total patient population for this part of the studywas defined as all patients who have met the eligibility criteria, notmet ineligibility criteria, and signed patient informed consent.

Some of the patients received a corticosteroid (e.g., dexamethasone)during the trial period.

It was assumed that the patients in the study had an overall failurerate of 0.45 per person-year of planned follow-up. It is approximately25% reduction in hazard rate compared to a hazard rate of 0.6 in thePhase III trial done by Stupp et al. With a total of 55 events among 76patients, the study yield 80% power to detect an observed hazard ratioof 0.75 (0.45 vs. 0.6) at an alpha level of 0.1(one-sided) to bestatistically significant. It yield above 90% power to detect a 30%reduction in hazard rate with observed hazard ratio of 0.7 (0.42 vs.0.6) at an alpha level of 0.1 to be significant. The overall failurerate was estimated by dividing the number of events (deaths) by thetotal exposure time in the study cohort along with 95% confidenceintervals. Survival probability and median time of survival wascalculated using Kaplan-Meier method.

Table 3 illustrates the demographics of the patients.

All Patients (N = 81) Age: Median (Range) 58 (27 - 80.9) Gender: No.Male (%) 51 (63) RACE: White: No. (%) 77 (95) Ethnic Group: 1 (1)Hispanic or Latino: No. (%) Not Hispanic or Latino: No. (%) 74(91)Unknown: No. (%) 6(7) Anticonvulsant: 64 (79) Yes: No. (%) No: No. (%)17 (21) KPS: 9 (11) 100: No. (%) 90: No. (%) 40 (49) 80: No. (%) 22 (27)70: No. (%) 8 (10) 60: No (%) 2 (2) Mini Mental Score: Median (Range) 29(22 - 30) Diagnosis: 80 (99) Glioblastoma Multiforme: No. (%)Gliosarcoma: No. (%) 1 (1) Surgical Procedure 77 (95) Craniotomy: No.(%) Biopsy: No. (%) 4 (5)

Table 4 illustrates toxicity and tolerability.

Adverse Events: N (%) Grade 3 Grade 4 Total Stupp et al, 2005 Acutekidney injury 1 (1) 1 (1) Alanine aminotransferase increased 1 (1) 1 (1)Anemia 2 (2) 2 (2) Aspartate aminotransferase increased 1(1) 1 (1)Atrial fibrillation 1 (1) 1 (1) Bronchial infection 1 (1) 1 (1)Cognitive disturbance 1 (1) 1 (1) Confusion 1 (1) 1 (1) Dehydration 1(1) 1 (1) Dizziness 1 (1) 1 (1) Dysphasia 1 (1) 1 (1) Fatigue 4 (5) 4(5) Flushing 1 (1) 1 (1) Generalized muscle weakness 2 (2) 2 (2)Headache 1 (1) 1 (1) Hyperkalemia 1 (1) 1 (1) Hypertension 1 (1) 1 (1)Hypokalemia 1 (1) 1 (1) Hypotension 1 (1) 1 (1) Hypoxia 1 (1) 1 (1)Lymphocyte count decreased 4 (5) 4 (5) Nausea 2 (2) 2 (2) NeutrophilCount Decreased 3 (4) 5 (6) 8 (10) 7% Platelet Count Decreased 4 (5) 11(13) 15 (18) 12% Rash maculo-papular 3 (4) 3 (4) Skin and subcutaneoustissue disorders 1 (1) 1 (1) Vomiting 1 (1) 1 (1) White Blood CellDecreased 5 (6) 3 (4) 8 (10) 7%

FIG. 3 and Table 5 show the overall survival analysis. Hazard rate ofdeath at 0.6 was the null hypothesis to against an alternativehypothesis of 0.42 by the trial design. The treatment has achieved thetarget therapeutic effect which yielded a hazard rate of 0.403 perperson year of follow-up.

TABLE 5 Trial Median Overall Survival Months, (95%CI) Hazard Rate (95%CI) (per person year of follow-up) ABTC0703 n=76 21.6 (16.1-23.7) 0.403(0.308-0.526) EORTC Stupp, 2005* n= 287 14.6 0.6 (*conversion) RTOG0525, 2013 STD arm, n=411* 16.6 0.501 (*conversion) RTOG 0525, 2013 DDarm, n = 422* 14.9 0.558 (*conversion)

FIG. 4 and Table 6 illustrate overall survival by MGMT status.

TABLE 6 Trial Median Overall Survival Months, (95%CI) Median OverallSurvival Months, (95%CI) MGMT mOS, MGMT methylated mOS, MGMTunmethylated ABTC0703 27 (n=29) 15.8 (n=37) EORTC Stupp, 2005 21.7(n=46) 12.7 (n=60) RTOG 0525 21.4 (n=122) sTMZ 20.2 (n=123) ddTMZ 14.6(n=254) sTMZ 13.3 (n=263) ddTMZ

FIG. 5 and Table 7 illustrate an increase in the percentage of patientswith 2 year survival and 3 year survival.

TABLE 7 Trial 2 Year Survival (% patients) 3 Year Survival (% patients)Iniparib Phase 2 N=76 42.1 23.7 EORTC Stupp, 2005 n= 287 27.2 16.0

Iniparib well tolerated at doses of 16 mg/kg weekly with radiation andTMZ and 17.2 mg/kg weekly with adjuvant TMZ

Single arm phase 2 met efficacy endpoint with at least a 25% reduced HRversus Stupp et al 2005

Also improved over RTOG 0525 (2013), but extrapolated and notpre-planned analysis.

Example 3 - Iniparib Is a Cytotoxic Anti-Tumor Prodrug Bioactivated byTrxR1/2 Materials

Cell line is purchased from the ATCC cell biology collection. Cellculture reagents are purchased from LifeTechnologies. All regularchemicals or reagents are obtained from Sigma-Aldrich Chemicals, unlessotherwise specified.

Cell Culture

HTB-16 cells are cultured in DMEM medium supplemented either with 10 %fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate and 10 µg/mlciprofloxacine (Euromedex) in a humidified 5% CO2 atmosphere at 37° C.

Fluorescence Microscopy.

HTB-16 cells are platted on polylysine D coated thin glass bottommicroscope chambers (Ibidi). After 24 h of culture cells are firstpre-incubated with BSO at 1 mM for 18 h and then treated with 100 µMiniparib-Biotin or its vehicle (DMSO 1%) for 30 min. For subcellularlocalization experiments mitochondria are stained with 100 nMMitotracker Red CMX (Molecular probes) added for 10 min. This stain isperformed before fixation (Paraformaldehyde 3.7 % in PBS pH 7.4). Biotinis developed, after Triton X100 (0.3% in PBS, 15 min) permeabilizationand saturation (1 % BSA + 1 % gelatin in PBS: saturation buffer), withAlexa-488 streptavidin (Molecular Probes) conjugate (1 µg/ml insaturation buffer). Nuclei are stained with Hoechst (Molecular Probes)and samples mounted in antifading solution (Ibidi).

Cells are imaged with a PLAN NeoFluar 40X (NA 1.3) or 100X, (NA 1.46)oil objectives on a LSM510 (Zeiss) confocal microscope. Laser lines,filters and dichroic mirrors are selected for maximal separation of thegreen (Ex./Em. 488/530 nm) and the red fluorescence (Ex./Em. 543/LP 585nm). Nuclei are observed (Ex./Em. 405/460 nm). For co-localizationstacks of images separated by 400 nm along z-axis are acquired.Post-capture processing is done using LSM510 software, stacks ofconfocal images were deconvoluted using the ImageJ software.

TrxR Activity

TrxR activity is measured by the reduction of 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) according to the manufacturer’sinstructions (TrxR assay kit, Sigma). Briefly, all incubations areperformed at 37° C. in 96-well microplates in 0.1 M potassium phosphate(pH 7.4), 10 mM EDTA and 240 µM NADPH. TrxR activity is measured byrecording the initial increase in A412 during the first 10 min uponaddition of 3 mM DTNB with a scanning multiwell spectrophotometer(Molecular devices). Endogenous TrxR activity is determined usingclarified octyl-glucoside cell lysates (50 µg proteins). Initialvelocities are derived from linear regression analyses and then plottedin double reciprocal plots to obtain the half time of TrxR inhibitionusing an one phase exponential decay analysis (Prism, GraphPadsoftware).

Trx Western Blot Analysis

A modification of a standard Western blot allows quantification of theredox state of specific proteins by separation of reduced and oxidizedforms by gel electrophoresis and detection of both forms with anantibody to an epitope that does not undergo oxidation-reduction (FIG. 6). Quantification is obtained directly from the relative intensities ofthe different bands. Two forms of the redox Western blot are developed,separating on the basis of differing charge (thiols are modified with acharged alkylating reagent) or mass (thiols are modified with ahigh-mass alkylating reagent). Redox analysis of the mitochondrialcompartment is performed using a redox Western blot analysis ofthioredoxin-2 (Trx2). Trx2 is exclusively found in mitochondria, so ananalysis of a cell or tissue extract provides specific information onredox in the mitochondria without fractionation. Analysis is performedfollowing derivatization with AMS. Trx2 contains two cysteine residues,and the addition of two molecules of AMS increases the mass byapproximately 1000 Da. Oxidized and AMS derivatized forms are separatedby nonreducing SDS polyacrylamide gel electrophoresis and detected byimmunoblotting.

ROS Production and Video-microscopy.

HTB-16 cells are platted in Ibidi® treated chambers. After 24 h ofculture cells are first pre-incubated with BSO at 1 mM for 18 h and thentreated with 100 µM iniparib or its vehicle (DMSO 1%) for 4 h. For ROSand nuclei detection, cells are respectively loaded with 5 µMCellROXOrange® (Molecular Probes) and 5 µM of DRAQ5® (Cell Signalingtechnology) in fresh media for 30 min.

Image acquisition is performed after washes with an Axiovert 200 Zeiss(Carl Zeiss Jena Germany) microscope equipped with a 40X C-Apochromatobjective (N.A.= 0.95). CellROXOrange® and DRAQ5® fluorescences arerespectively excited with a LED light source (595 and 646 nm) andemitted light are collected at 565 and 681 nm. For quantification ImageJsoftware is used. Data is presented as Integrated Intensities/nuclei.

Clinical Trial Panel.

Clinical trials are conducted to test the efficacy of iniparib forpatients with glioblastoma. Formalin-fixed, paraffin-embedded (FFPE)archival samples from biopsy or surgery at original diagnosis ofglioblastoma are profiled on the exon-based Affymetrix Hugene1.0STmicroarrays using an RNA extraction protocol adapted to the shortfragment lengths resulting from RNA degradation in FFPE samples.

Processing of Gene Expression Data.

The raw gene expression data in the form of individual Affymetrix CELfiles are processed using MAS5 estimation. Quality control based onaverage array brightness further excludes outlier scans, and theremaining profiles are then normalized to each other using quantilenormalization. Affymetrix probesets are mapped to genes, in a mannerwhere multiple probesets mapping into the same gene are resolved byassigning the highest intensity to the corresponding gene. The data isthen log2-transformed, and standardized by mean-subtraction and divisionby the standard deviation across all samples, for each gene separately.Finally, the data matrix is subsetted to the intersection of theoxidative response set of genes with the set of genes represented on theHugene1.0ST arrays.

Multivariate Cox Modeling, Based on Gene Expression Data and on an aPriori Oxidative Stress Gene Set.

A multivariate Cox model using supervised principal components is usedto model progression free survival times regressed on gene expressiondata. The model is of the form

$\xi = \log\left( \frac{\lambda\left( {t\left| {x,z} \right)} \right)}{\lambda_{0}(t)} \right) = \beta_{0}z + {\sum\limits_{l = 1}^{K}{{\widetilde{\beta}}_{r}{\widetilde{x}}_{l} + z \cdot {\sum\limits_{i = 1}^{k}{{\widetilde{\gamma}}_{l}{\widetilde{x}}_{l}}}}}$

where by definition ξ is the “log-hazard-ratio” for a given individual,λ(t|z, x) the hazard function (or risk per unit time) for thatindividual, with covariate vector (z, x), and PFS time t, λ0(t) thebaseline hazard function (the hazard which applies to an individual withall covariates exactly equal to 0), and where z is a binary indicator oftreatment arm, with z = 0 for the control and z = 1 for the iniparibtreatment arm. The symbol x refers to the gene expression vector with p= a specific set of components (the subset of the oxidative stress geneset represented on the Affymetrix microarrays). Note that this modelcontains both direct and interaction terms, the coefficients β̃₁accounting for the direct effects of gene expression (which might becalled “prognostic” effects) and the coefficients γ₁ accounting for geneexpression × treatment-arm effects (“predictive” effects). Thecoefficient β0 accounts for overall, gene-expression independent,treatment-arm effects.

While preferred embodiments of the present disclosure have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the disclosure. It should beunderstood that various alternatives to the embodiments of thedisclosure described herein may be employed in practicing thedisclosure. It is intended that the following claims define the scope ofthe disclosure and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

1-30. (canceled)
 31. A method of treating glioblastoma in a humansubject in need thereof, the method comprising: administeringconcurrently to the subject (i) of 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof, and (ii) temozolomide, and furtheradministering radiation.
 32. The method of claim 31, wherein about 5milligrams per kilogram (mg/kg) to about 10 mg/kg of the4-iodo-3-nitrobenzamide or the salt, metabolite or prodrug thereof isadministered to the human subject.
 33. The method of claim 32, whereinthe 4-iodo-3-nitrobenzamide or the salt, metabolite or prodrug thereofis administered to the subject in an amount of about 8 mg/kg.
 34. Themethod of claim 32, wherein the 4-iodo-3-nitrobenzamide or the salt,metabolite or prodrug thereof is administered to the subject twice aweek.
 35. The method of claim 34, wherein the 4-iodo-3-nitrobenzamide orthe salt, metabolite or prodrug thereof is administered to the subjectfor about six weeks.
 36. The method of claim 31, wherein 75 milligramsper square meter (mg/m²) to about 200 mg/m² of the temozolomide isadministered to the subject.
 37. The method of claim 36, wherein 150milligrams per square meter (mg/m²) to about 200 mg/m² of thetemozolomide is administered to the subject.
 38. The method of claim 36,wherein temozolomide is administered to the subject daily.
 39. Themethod of claim 36, wherein temozolomide is administered to the subjectfor about six weeks.
 40. The method of claim 31, wherein about 60 grays(Gy) of total radiation is administered to the subject.
 41. The methodof claim 40, the radiation is administered to the subject over a courseof about six weeks.
 42. The method of claim 31, further comprising amaintenance regimen comprising administering (i) 4-iodo-3-nitrobenzamideor the salt, metabolite or prodrug thereof, and (ii) temozolomide. 43.The method of claim 42, wherein about 5 mg/kg to about 10 mg/kg of4-iodo-3-nitrobenzamide or the salt, metabolite or prodrug thereof isadministered to the subject during the maintenance regimen.
 44. Themethod of claim 43, wherein about 8.6 mg/kg of the4-iodo-3-nitrobenzamide or the salt, metabolite or prodrug thereof isadministered to the subject during the maintenance regimen.
 45. Themethod of claim 43, wherein the 4-iodo-3-nitrobenzamide or a salt,metabolite or prodrug thereof is administered to the subject twice aweek.
 46. The method of claim 42, wherein about 75 mg/m² to about 200mg/m² of the temozolomide is administered to the subject during themaintenance regimen.
 47. The method of claim 42, wherein the4-iodo-3-nitrobenzamide or the salt, metabolite or prodrug thereof andthe temozolomide is administered to the subject for about 1-6 cycles,wherein a single cycle is about 28 days.
 48. The method of claim 47,wherein the temozolomide is administered to the subject on Days 1-5 ofeach cycle.
 49. The method of claim 31, wherein the glioblastoma is aprimary glioblastoma or a secondary tumor.
 50. The method of claim 31,wherein the subject has a grade III or grade IV glioblastoma.